Ординатура / Офтальмология / Английские материалы / Ocular Periphery and Disorders_Dartt, Bex, Amore_2011

intracellular contractile mechanisms that rip the trailing focal adhesions from the underlying stratum, sometimes even leaving part of the cell membrane behind. This cycle allows the cells to loosely adhere to the underlying matrix so that they can move due to contraction and reformation of the actin cytoskeleton.

The third phase of corneal wound healing is the proliferation/restratification phase. During this phase, the basal cells which have just finished the migratory phase and were kept in a nondividing state begin to proliferate once more in order to repopulate then differentiate and restratify the corneal epithelial layers and smooth out any irregularities in the BM. Also during this phase, the epithelial basement membrane (EBM) is reformed (in penetrating or large debridement wounds), the provisional matrix is broken down, and permanent attachments are reformed between the basal epithelial cells and the EBM. In nonpenetrating wounds, this phase is usually accomplished within a few weeks. In penetrating wounds, remodeling of the ECM may persist for months and even years after the insult, and may never completely regenerate.

Corneal Nerves

The cornea is one of the most densely innervated tissues of the body and corneal nerves have been shown to play a significant role in the maintenance of the cornea and in various corneal diseases. There are three networks of corneal nerves that enter the stroma and innervate the mid-stromal, bowman’s layer, and epithelial layers. Regeneration of epithelial nerves has been shown to depend on the type of insult sustained. For example, reinnervation of the epithelial layer is much faster following laser ablation of the epithelium versus manual debridement and reinnervation density correlates with renewed corneal sensitivity. Following penetrating wounds, as in a laser-assisted in situ keratomileusis (LASIK) procedure, nerve bundles in the Bowmans layer and stromal flap (those that were severed) disappear completely. Within the first 6 months, nerves first reinnervate the stromal flap, then the Bowman’s layer. A cycle of regeneration has been observed where the number of nerves is brought back to normal levels by 2 years post-LASIK, then reduced significantly by 3 years out though the reason and mechanism of this loss remains unknown. Nerve densities in the stroma below the wound remain unchanged following wounding.

Although there is much greater complexity involved in penetrating wound healing scenarios, including the drastic alteration of the stromal keratocyte phenotype with the possibility of permanent scarring, the overall phases of the penetrating corneal wound healing (i.e., lag, migration, and proliferation/restratification) is similar to those of the nonpenetrating process. Important additional differences include cell invasion from limbal

blood vessels, the greater cytokine expression profile, and the much longer time frame for remodeling of the wound microenvironment due to the greater scale of remodeling that must take place in the stroma in order to maintain the optimal arrangement for light transmission.

Corneal Epithelial Wound Healing:

Cell–Cell and Cell–Matrix Junctions

Cell–cell and cell–matrix junctions are critical components of both unwounded and wounded cell environments and every tissue in the body has a unique profile. Along with secreted factors, these junctions are responsible for all the possible signaling triggers that can alter gene expression and affect cell phenotype, function, and survival. Additionally, these junctions in combination with cellular cytoskeletons give a tissue its structure and help define function. Following wounding, some of these junctions must be broken down while others must be formed to allow cells to migrate while still others must remain to retain as much of a barrier to the outside environment and ensure a cohesive, coordinated sheet of cells resurfaces the wound rather than cells migrating individually. There are four known types of cell–cell interactions: gap junctions, tight junctions, adherins junctions, and desmosomes.

Cell–Cell Junctions: Gap Junctions

Gap junctions are formed by connexins which are homoor heterohexameric proteins that form on the lateral side of cells and can form both homoand heterotypic interactions with each other in order to connect the cytoplasm of two neighboring cells between an intercellular space. Gap junctions allow the passage of low- molecular-weight proteins (water, ions, secondary messengers, electrical impulses, and low-molecular-weight (<1 kDa) metabolites and nutrients) to pass freely from one cell to another, allowing cells to communicate quickly with each other in order to coordinate such physiological processes as development and regeneration.

Connexins(cxs) are named according to their molecular weight; thus, connxin43 is a 43-kDa protein. Multiple connexins have been observed in the cornea by studying various mammalian species (Table 1). Connexin 43 is the most well-documented connexin in the corneal epithelium and it is found primarily in the basal epithelial cells with expression decreasing progressively toward the superficial layers. Other connexins such as cxs26, 30, 31.1, 37, and 50 have been observed with spatially distinct patterns in the unwounded cornea. No connexins have been detected in migrating CECs following wounding though gap junctions in cells distal to the wound remain; thus, gap junctions in cells proximal to the wound are broken down during the lag phase and expression is downregulated throughout the

Table 1 Cell–cell junctions in the unwounded and wounded cornea

migration phase. Following resurfacing, gap junction reformation correlates with the reappearance of laminin-1 in the EBM. Expression of connexin43 has been attributed to Rho, but not ROCK signaling. The functional significance of the loss of gap junctions in migrating cells has yet to be determined.

Cell–Cell Junctions: Tight Junctions

Tight junctions are homeotypic interactions by transmembrane protein structures which link the actin cytoskeleton of neighboring cells and act like cellular gaskets. These structures are composed mainly of claudin occludin and junctional adhesion molecule A ( JAMA) and have several purposes: they link neighboring cells, separate proteins in the apical cell membrane from those in the basal–lateral, and they prevent their passage of ions and other small molecules from penetrating between neighboring cells, thus contributing to corneal deturgesence. In the cornea, tight junctions have been detected in the superficial and wing cells. Early work in the field had found that tight junctions were reduced at the wound margin following wounding but reformed quickly behind the migrating front of epithelia. However, more recently, it has been shown that these junctions persist following corneal epithelial ablation with no significant alteration in expression. This newer finding correlates well with an older finding that showed a component of the tight junction complex, ZO-1, is upregulated following removal of superficial CECs. While there is no direct evidence yet,

retention of tight junctions presumably helps ensure the integrity of the corneal surface as much as possible and helps to keep cells migrating together as a cohesive sheet.

Cell–Cell Junctions: Adherens Junctions

Adherens junctions are also found on the lateral side of cells and keep neighboring cells firmly attached to each other by anchoring to the actin cytoskeleton. Adherens junctions in the cornea are formed by homeotypic dimers of transmembrane E-cadherin molecules which bind each other in the intercellular space. E-cadherin is calcium dependent and is anchored intracellularly to the actin cytoskeleton by vinculin and a- and b-catenin molecules. E-cadherin-containing adherens junctions have been found throughout the cornea epithelium, and wounding does not seem to affect the expression or distribution of these junctions. Similar to tight junctions, adherens junctions may be retained in order to keep cells migrating together as a cohesive sheet, though again, direct proof is still lacking.

Cell–Cell Junctions: Desmosomes

The final type of cell–cell junction is the desmosome. Desmosomes are similar to adherens junctions in that they both function to keep neighboring cells attached to each other and they are both composed of molecules from the cadherin family. However, unlike adherens junctions which are composed of the cadherin family member E-cadherin, desmosomes are composed mainly of the caherin family members desmoglein and desmocollin. Also, while the adherens junction is anchored to the actin cytoskeleton, desmosomes are anchored by desmoplakin, plakoglobin, and plakophillin to the intermediate filament (IF), or cytokeratin, cytoskeleton. Interestingly, unlike adherens junctions, desmosomes do alter their expression pattern following wounding. It has been found that similar to connexin43, desmoglein is downregulated in migrating CECs, and this downregulation persists until resurfacing is complete and laminin-1 reappears below the epithelial cells. Like gap junctions, the functional significance of the loss of desmosomes in migrating cells has yet to be determined, though it may be necessary for the morphological changes needed for migration.

Cell–Matrix Junctions: Hemidesmosomes

and Adhesion Complexes

In addition to cell–cell interactions, cell–matrix interactions are also critical components of tissue function and structure in normal, wounded, and diseased tissue, and a lack of adherence to an underlying matrix may result in apoptosis by ceratin cell types (such as epithelia) and a separation of the dermal and epidermal layers. Cells can be connected to an underlying extracellular matrix by

adhesions that are anchored intracellularly to either the actin or IF cytoskeleton, both of which utilize integrins to form these anchors.

Integrins are not only structural proteins which affect cell attachment, but they are also potent signaling molecules that can affect cell proliferation, migration, and survival properties. Intergins form heterodimers of an a- and b-chain and the different combination of chains determines the extracellular binding partner; some have only one substrate while others have multiple substrates with differing binding kinetics. In mammals, there are 19 known a-chains and 9 known b-chains which can assemble into about 25 distinct integrins. Integrins a2b1, a3b1, a6b1, a9b1, avb1, avb5, avb6, and a6b4 have all been localized to the corneal epithelium with the strongest expression being found in the basal epithelial cells and getting progressively weaker as the cells get closer to being sloughed off into the tear fluid. This progressive loss of integrins ensures that cells that migrate away from the basal epithelium proliferate less and provide a less adhesive surface for microbial adherence and infection of the corneal surface.

Hemidesmosomes are present at the basal membrane of unwounded basal epithelial cells and are composed of the a6b4 integrin as well as adaptor proteins for linking this protein to the IF cytoskeleton. In the unwounded cornea, hemidesmosomes are tightly bound to anchoring fibers composed of collagen VII which pass perpendicular through the BM then make a 90 turn and wrap around collagen III bundles. Following wounding, hemidesmosomes are broken down and are no longer detectable for 70–200 mm outside the wound area in order to allow cells to begin the migratory phase. Although the hemidesmosomes are absent, a6b4 integrin expression is actually increased during resurfacing, though these integrins are no longer associated with hemidesmosomes but instead form focal adhesion complexes (discussed earlier) which now connect the components of the underlying provisional matrix such as unprocessed laminin-5 with the actin cytoskeleton. In penetrating wounds, reformation of the basal lamina and hemidesmosome complexes occurs concurrently in about 6–8 weeks, though studies in monkeys have shown that reformation of the anchoring fibrils may take as long as 18 months to reform. All of these time frames are approximations and can be affected by variables such as the size of the wound, the age and species of the subject, and if there are any complicating factors such as infection or genetic abnormalities.

Secreted Factors Involved in

Corneal Epithelial Wound Healing

Along with cell–cell and cell–matrix junctions, secreted factors such as cytokines, growth factors, and proteases are also responsible for regulating normal tissue maintenance

and wound healing properties. Cytokines and growth factors are small molecules that can bind and activate their cognate cell surface receptors. These activated receptors then transduce the signal to the nucleus via various intracellular signaling pathways such as the mitogen-activated protein kinase (MAPK), Smad, and nuclear factor kappa-light- chain-enhancer of activated B cells (NF-kB) pathways. However, signaling is not necessarily linear and cross-talk between the pathways can and often does occur.

In other epithelia of the body, these factors are usually secreted by platelets or other cell types that enter a wound from the blood stream. The cornea, however, is an avascular tissue and thus all signals must come from either the tear fluid or the epithelial and stromal cells themselves. Corneal epithelial and stromal cells have been shown to secrete a host of cytokines and growth factors. Transcriptional analysis of primary corneal epithelial cultures has found insulin-like growth factor (IGF), epidermal growth factor (EGF), transforming growth factor (TGF)-a, b-fibroblast growth factor (FGF), TGF-b1, TGF-b2; in addition, all of their cognate receptors are expressed by both corneal epithelial and stromal cells, though to varying degrees, and may act via both autocrine and paracrine mechanisms. Some other important factors have more specific localization patterns and generally act via paracrine signaling. For example, both keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) are only expressed by stromal keratocytes though their cognate receptors are much more preferentially expressed by epithelial cells, while plateletderived growth factor (PDGF)-bb is expressed by epithelial cells and its cognate receptor is only expressed in stromal cells. The inflammatory cytokines interleukin (IL)-1a and IL-1b are only expressed by epithelial cells in the unwounded state, but their cognate receptor is present on both epithelial and stromal cells. However, stimulation of keratocytes by IL-1 as occurs during penetrating wounds can induce an autocrine feedback loop; thus, keratocytes can express IL-1. Furthermore, this IL-1 feedback loop also induces other secreted factors such as HGF and KGF expression by keratocytes.

Cytokines can induce gene expression changes that can alter the proliferation, migration, and survival properties of a cell. For example, it has been shown that KGF and EGF can induce CEC proliferation. Also, while neither EGF, PDGF-bb, IL-1, nor TNF-a alone can induce epithelial cell migration, when combined with fibronectin as a substrate, these factors can all significantly speed up migration rates as compared to fibronectin alone. This further emphasizes the complex and combinatorial signaling that controls corneal epithelia wound healing. The role of the TGF-b family in epithelial migration is controversial with some labs showing that it enhances epithelial migration rates, and others finding that it slows down resurfacing rates. Further studies must be done to reconcile these seemingly opposite results.

Matrix Metalloproteinases

Matrix metalloproteinases (MMPs) are a family of structurally related zinc-dependent proteases with overlapping substrate specificities. MMPs are known regulators and effectors of many cellular processes, including tumor progression, development, and epithelial wound healing as well as normal tissue maintenance. The MMP family consists of over 20 members whose substrates have come to include all extracellular membrane proteins as well as many cell surface proteins (i.e., cadherins, heparin-binding EGF (HB-EGF), etc.) and secreted proteins (i.e., TGF-b, IL-1, TNF-a, etc.) which can be both activated and degraded by MMPs. Thus, MMPs can affect gene expression by alterating intracellular (nuclear) signaling by cleavage of cell–cell and cell–matrix interactions as well as by altering the active cytokine and growth factor microenvironment. Due to their broad substrate specificity and potent proteolytic activity, MMPs must be tightly controlled, and are regulated at the transcriptional and posttranslational levels. MMPs can be either secreted or membrane bound and contain a propeptide at their N-terminal that must be removed before they can become proteolytically active.

MMPs are critical components of the wound healing process, as global MMP inhibition by pharmacological agents results in a failure to resurface a wound. Similarly, an overexpression of MMPs is also involved in many epithelial disorders such as idiopathic pulmonary fibrosis in the lung, dystrophic epidermolysis bullosa in the skin, and diabetic retinopathy and recurrent corneal erosion in the eye. Therefore, a precise level of MMP expression is necessary for normal tissue maintenance and optimal wound repair.

In the unwounded cornea, most MMPs are expressed at basal or undetectable levels, except for MMP-7 and -14 which are expressed constitutively. Following removal of the epithelium, several MMPs are upregulated and various family members take on a distinct localization pattern, though the expression patterns of MMP-7 and -14 do not change significantly. MMP-9 is the most well-studied family member and is strongly upregulated at the very tip of the migrating epithelial cells. MMP-10 becomes upregulated by all migratory cells, and MMP-13 expression is upregulated by cells throughout the wound area and in the periphery of the wound. MMP-12 mRNA upregulation has also been detected in the peripheral cells, but this upregulation has not been detected for the actual protein. Penetration of the EBM induces expression of more MMPs such as MMP-2, -3, and possibly -8 which are used to remodel the stromal compartment.

Following resurfacing of nonpenetrating wounds, MMP levels are generally decreased, though MMP-9 expression persists and spreads distally from the wound closure with the timing of its expression correlating with degradation of

the provisional matrix. This correlation has been confirmed using MMP-9 knockout (KO) mice which are unable to degrade the fibrin in the provisional matrix which remains as a corneal haze. However, a recent study in human eyes has found that people displaying uncomplicated LASIK surgeries (penetrating wound) may, up to 7 years later, still have EBM irregularities around the wound edges that lead to stromal–epithelial contact causing MMP expression and indicating a continuing wound healing process.

While MMP-9 is critical for overall corneal wound regeneration, it is not critical for corneal re-epithelialization, and in fact acts to slow the rate of re-epithelialization. Other MMPs, however, are critical for corneal resurfacing as global inhibition of MMPs retards resurfacing. However, which specific MMPs are necessary, what their substrates are, and why these MMPs are necessary is still unknown. These questions are especially difficult to answer because loss of an MMP, as in a knockout or knockdown model, can be compensated for by other MMPs due to the overlapping substrate specificities as indicated by the low number of MMP KO mice displaying drastically different phenotypes.

Corneal Epithelial Wound Healing:

Conclusion

While the corneal epithelial wound healing process shares some similar properties with skin, lung, and gut epithelial wound healing, there are important differences. For example, whereas skin epithelia can heal by a scarring or vascularization process, the corneal wound healing process must minimize these parameters in order to retain optimal visual clarity. Formation of scar tissue on the molecular level means the accumulation of a-smooth muscle actin-containing fibroblasts also known as granular tissue myofibroblasts that form the fibrous tissue in the early scar before the secretion and formation of the older collagen scar tissue. As the central cornea is an avascular tissue, formation of a-smooth muscle actin expressing cells is initially derived solely from stromal keratocytes. Stromal keratocytes are induced to differentiate into a-smooth muscle actin expressing cells by secreted factors expressed by the CECs and present in the tear fluid such as TGF-b. In the unwounded eye, the EBM binds TGF-b and acts as a physical barrier to prevent the stromal keratocytes from being activated. Over a remodeling period of weeks, months, or even years, myofibroblasts can slowly disappear from a wound site via an unknown mechanism, possibly apoptosis. This disappearance is by no means guaranteed and even if it does occur, it may or may not improve visual acuity depending on whether or not a collagenous scar tissue has formed.

The severity of the wound response is correlated with the severity of the wound; wounds that do not penetrate the EBM tend to regenerate completely, whereas wounds

that do penetrate the EBM tend to induce myofibroblast differentiation, require much longer to heal, and may never quite regain the original architecture. Thus, the most important factor involved in corneal wound healing is whether or not the EBM has been disrupted. While penetration of the BM is arguably the most important factor in corneal wound healing, it is by no means the only determining factor; the size of the wound, the age of the subject, and the causative agent are also important factors. Additionally, any defect in the tear fluid composition or integrity may further impair the wound healing process. All of these factors can affect the cellular microenvironment by alteration of cell adhesion molecules (CAM) and secreted factors which both coordinate and drive the wound healing process.

See also: Corneal Epithelium: Transport and Permeability; Corneal Nerves: Anatomy; Refractive Surgery and Inlays.

Further Reading

Fini, M. E., Cook, J. R., and Mohan, R. (1998). Proteolytic mechanisms in corneal ulceration and repair. Archives of Dermatological Research

290(supplement): S12–S23.

Fini, M. E. and Stramer, B. M. (2005). How the cornea heals: Corneaspecific repair mechanisms affecting surgical outcomes. Cornea 24 (8 supplement): S2–S11.

Gipson, I. K., Spurr-Michaud, S. J., and Tisdale, A. S. (1988). Hemidesmosomes and anchoring fibril collagen appear

synchronously during development and wound healing.

Developmental Biology 126(2): 253–262.

Imanishi, J., Kamiyama, K., Iguchi, I., et al. (2000). Growth factors: Importance in wound healing and maintenance of

transparency of the cornea. Progress in Retinal and Eye Research

19(1): 113–129.

Li, D. Q. and Tseng, C. G. (1995). Three patterns of cytokine expression potentially involved in epithelial–fibroblast interactions of human ocular surface. Journal of Cell Physiology 163(1): 61–79.

Lu, L., Reinach, P. S., and Kao, W. W.-Y. (2001). Corneal epithelial wound healing. Experimental Biology and Medicine (Maywood)

226(7): 653–664.

Mohan, R., Chintala, S. K., Jung, J. C., et al. (2002). Matrix metalloproteinase gelatinase B (MMP-9) coordinates and effects epithelial regeneration. Journal of Biological Chemistry 277(3): 2065–2072.

Schultz, G. S., White, M., Mitchell, R., et al. (1987). Epithelial wound healing enhanced by transforming growth factor-alpha and vaccinia growth factor. Science 235(4786): 350–352.

Sivak, J. M. and Fini, M. E. (2002). MMPs in the eye: Emerging roles for matrix metalloproteinases in ocular physiology. Progress in Retinal and Eye Research 21(1): 1–14.

Steele, C. (1999). Corneal wound healing: A review. Part I. Optometry Today 24: 28–32.

Stepp, M. A. (2006). Corneal integrins and their functions. Experimental Eye Research 83(1): 3–15.

Stepp, M. A., Spurr-Michaud, S., and Gipson, I. K. (1993). Integrins in the wounded and unwounded stratified squamous epithelium of the cornea. Investigative Ophthalmology and Visual Science 34(5):

1829–1844.

Stramer, B. M., Zieske, J. D., Jung, J. C., Austin, J. S., and Fini, M. E. (2003). Molecular mechanisms controlling the fibrotic repair phenotype in cornea: Implications for surgical outcomes.

Investigative Ophthalmology and Visual Science 44(10): 4237–4246. Suzuki, K., Saito, J., Yanai, R., et al. (2003). Cell–matrix and cell–cell

interactions during corneal epithelial wound healing. Progress in Retinal and Eye Research 22(2): 113–133.

Zieske, J. D. (2001). Extracellular matrix and wound healing. Current Opinion in Ophthalmology 12(4): 237–241.

Glossary

Barrier – Protein-containing formations between cells in the more superficial layers of the corneal epithelium to prevent infiltration of pathogens into the underlying stroma.

Deturgescence – Physiological state of corneal hydration required for optimal transparency.

Electrochemical equilibrium – Membrane voltage and intracellular ionic activity at which there is no net flux of a specific ion across a membrane.

Net flux – Algebraic difference between unidirectional ionic fluxes across a membrane, which is indicative of active ion-transport activity.

Transient receptor potential protein – Membrane proteins in a superfamily of 27 different genes that arrange themselves in tetrameric configurations to form plasma membrane channels, which can be activated by a large variety of stimuli.

Introduction

Corneal epithelial ion transport and permeability underlie the ability of this tissue to maintain corneal transparency. In this article, we describe how different types of receptors modulate through second messenger-signaling control of ion transporter function and cell membrane permeability. Figure 1 depicts a schematic summarizing the current picture of ion transporters and channels mediating control of cornea epithelial renewal and tissue transparency. To provide insight into the outcome of dysregulation of transporter activity and permeability, we discuss strategies that may circumvent these pathophysiological consequences.

The cornea provides about 75% of the total refractive power required for normal vision. This function depends on the ability of the cornea to remain transparent. The corneal epithelial layer undergoes continuous renewal every 14–28 days. This process preserves its integrity and assures that the cornea is protected from environmental pathogenic infiltration. Such protection is provided by tight junctional continuity forming a moderately high resistance barrier between neighboring cells. This resistance is essentially only selectively permeable to low-molecular-weight

ionic species. As the epithelial top layers continuously undergo terminal differentiation followed by being sloughed off, the maintenance of tight junctional integrity is critical for the corneal epithelial to provide its barrier function. Any disruption of upper cell layer apposition that is not rapidly repaired through wound healing renders the cornea vulnerable to infection, swelling, and opacification leading to losses in visual acuity. Losses in tissue transparency occur since swelling of the corneal ground substance leads to disruption of extracellular matrix organization and an irregular corneal surface. These changes cause impinging light to be reflected or scattered, instead of being refracted. Therefore, studies directed toward understanding how the corneal undergoes continuous renewal are relevant for delineating strategies to restore corneal transparency in a clinical setting.

Corneal Hydration Control and

Transparency

In humans, the cornea is approximately 500–550 mm thick and is composed of three layers: epithelium, stroma, and endothelium. These tissues have different embryonic origin. Corneal thickness is dependent on the hydration state of the stromal ground substance, whose physiochemical properties cause continuous fluid imbibition. Excessive fluid uptake by the stromal ground substance lying between collagen lamellae results in corneal swelling and opacity.

The stromal ground substance continuously imbibes fluid from the tears and the aqueous humor in the anterior chamber facing the endothelium. This imbibing process is equivalent to a negative pressure of approximately60 mmHg. In order for this suction effect not to lead to excessive swelling, it is essential that the corneal epithelium, in concert, with the underlying endothelial layer mediate osmotically coupled water flow outward from the stroma through net ion transport. The endothelial layer facing the anterior chamber provides most of the dehydrating function whereas the epithelial layer contribution is relatively minor. In other words, epithelial layermediated net fluid transport toward the tears from the stroma appears to play a fine-tuning role in maintaining a state of corneal hydration commensurate with normal vision. Nevertheless, under stimulated conditions, the rabbit epithelial layer can provide up to 25% of the total

H+

K+

Na+

Tears

Na+

H+

Cl−

Apical membrane Tight junction

Ca2+

Basolateral

membrane

Ca2+

K+ 2K+

Na+ 3Na+

Cl−

HCO−3

Cl−

Paracellular

dehydrating function. However, if the epithelial layer integrity is damaged, fluid leakage from the tears cannot be compensated by the dehydrating function of the innermost endothelial layer. In this case, the stroma will continuously swell. Therefore, epithelial wound healing through cell proliferation, migration, and tight junction renewal is crucial for maintaining the corneal barrier function.

Importance of Transport Mechanisms to Epithelial Function

The corneal epithelial electrical resistance is dependent on the intactness of its tight junctions forming appositions in the upper layers of the epithelial functional syncytium. In order to provide an effective barrier function, the electrical resistance of the corneal epithelium has to be relatively high (i.e., at least 1 kO cm2). In addition, the ability of the epithelium to mediate net ion transport from the stroma into the tears is dependent on tight junctional resistance. If the resistance falls due to injury, there is a corresponding decrease in net ion transport, resulting in compromise of the barrier function followed by a decline in osmotically coupled fluid flow toward the tears.

In all species studied, the corneal epithelial layer elicits secondary active chloride (Cl ) transport from the stroma to the tears whereas net sodium (Na+) transport occurs in the opposite direction, from the tears to the stroma. There is variability in the magnitude of active Na+ transport due to differences in the Na+ permselectivity of the tear-side- facing apical membrane. In rabbits and humans, the relative Na+ and Cl permeabilities are similar to one another whereas in the amphibian this membrane is essentially Cl permselective. There is large variability among different species with regard to the ratio between tear-directed Cl transport and stroma-directed Na+ transport. In the rabbit and human, this ratio under baseline conditions is about 50%, whereas in the amphibian (i.e., toad and bullfrog) it is at least 90%. Despite such differences, osmotically coupled fluid flow across this layer has been identified both in the isolated rabbit and amphibian cornea suggesting that net Cl fluxes toward the tears exceed stroma-directed Na+ fluxes.

Corneal transparency is dependent on net influx and efflux of inorganic and organic osmolytes. For example, amino acid uptake mechanisms have been identified in this tissue. By definition, an active uptake mechanism depends on metabolic energy generated by aerobic and anaerobic metabolism. Its metabolic dependence is evident, since exposure to metabolic inhibitors results in

corneal swelling and the development of opacification. These changes occur because corneal deturgescence (i.e., physiological hydration state) and maintenance of epithelial health are dependent on the ability of this tissue to: (1) elicit osmolyte and drug extrusion against opposing electrochemical gradients and (2) accumulate organic substrates to levels above those in the extracellular bathing solution. This array of very different transport functions are performed by transporters of distinct ionic and substrate selectivity.

Primary and Secondary Ionic Transport

Mechanisms

The corneal epithelial cells express both primary and secondary ionic transport mechanisms. As in all epithelial cells, Na+:K+ ATPase expression occurs along the basolateral membrane facing the stroma and the paracellular pathway between the neighboring epithelial cells. This transport mechanism is primary since its ATPase activity directly elicits electrogenic Na+ extrusion into the stromal and paracellular media. Such efflux is coupled to intracellular K+ accumulation above its predicted electrochemical value. On the other hand, secondary ionic transporter function is driven by the Na+ and K+ electrochemical gradients established by Na+:K+ ATPase activity. In all species, there is Na+:K+:2Cl co-transport activity in the basolateral membrane, which is reflective of secondary active ion transport function. This cotransporter functions to accumulate intracellular Cl to levels above its predicted electrochemical values. Net Cl transport into the tears occurs as a consequence of its efflux by electrodiffusion across the tear-side facing essentially Cl permselective apical membrane. In order to maintain charge neutrality, Na+ moves in parallel as a counter ion through the paracellular pathway.

Another primary active ion transporter identified in the corneal epithelium is the H+ pump. Its activity in combination with two different types of cotransporters is required for mediating regulation of intracellular pH. These cotransporters are Cl :HCO3 and Na+:H+ antiporters whose transport directionality is determined by the electrochemical gradients of the involved ions. Specifically, the Cl :HCO3 antiport will drive HCO3 out of the cell, provided there is sufficient carbonic anhydrase activity to raise intracellular bicarbonate levels above those in the external medium. Under such conditions, Cl will be taken up from the stroma into the cell interior in exchange for bicarbonate efflux into the external medium. In this scenario, intracellular pH falls resulting in increases in the activity of two different alkalinizing mechanisms: Na+:H+ antiport activity in combination with H+ pump ATPase activity. The Na+:H+ antiport can provide an alkalinizing function if the Na+:K+ pump

activity is sufficient to establish a steep enough Na+ gradient between the external milieu and the intracellular compartment to exchange H+ from the cell interior for downhill Na+ movement into the cell interior.

Cl efflux out of the epithelial cells across the apical membrane is dependent on membrane voltage electronegativity relative to the bathing solution. Numerous studies have shown that the intracellular membrane voltage is about 50 mV. This value is dictated to a large extent by the activity of the electrogenic Na+:K+ pump and the relative K+ permeability of the basolateral membrane. The electrogenic Na+:K+ exchange pump contributes since its pump Na+:K+ stoichiometry exceeds a value of 1. In other words, with each pump cycle, more Na+ ions are extruded than K+ ions are taken up into the cell interior from the stroma. In any case, due to the activity of the Na+:K+ pump, Cl is accumulated into the epithelial cells above its predicted electrochemical equilibrium value. In turn, K+ diffuses downward and outward, essentially across the basolateral rather than the apical membrane since the former barrier has a much higher K+ permselectivity. This downward movement of K+ establishes a negative membrane voltage which is dependent on the activity of the Na+:K+ pump and the relative basolateral membrane K+ permeability. Another factor affecting the level to which K+ is elevated above electrochemical equilibrium is the tightness of coupling between the Na+:K+:2Cl cotransporter and the Na+:K+ pump. Stimulation of the Na+:K+:2Cl cotransporter may occur as a consequence of increases in Na+: K+ pump activity leading to enhancement of intracellular K+ accumulation. A rise in K+ can then be compensated for by a rise in basolateral K+ membrane permeability, which will increase outward directed K+ efflux and the membrane voltage electronegativity. This chain of events will stimulate net Cl efflux from the stroma into the tears since the driving force for Cl efflux into the tears, across the highly Cl permselective apical membrane, is the magnitude of the membrane voltage electronegativity.

Therefore, it is through the concerted activity of the aforementioned transport mechanisms, along with appropriate regulation of the cell-limiting membranes, that the corneal epithelium can elicit net Cl transport from the stroma toward the tears and regulate its intracellular pH within bounds that are required for the maintenance of anterior ocular surface health.

Coupling of Ionic Transport Mechanisms to Stromal Deturgescence

It is commonly thought that the driving force for fluid transport out from the stroma across the epithelial cells into the tears is local osmosis. The fact that the corneal epithelium elicits net Cl transport into the tears suggests

that this ionic flux may be sufficient to account for the magnitude of outward-directed fluid transport. This expectation has been proven to be correct since, under open circuit conditions, net Cl transport is commensurate with measured isotonic fluid transport. The dependence of transepithelial fluid transport into the tears on net Cl transport was validated by showing that drugs which either stimulate or inhibit net Cl transport have corresponding effects on fluid transport.

The fact that there is a coupling between fluid and net Cl transport prompted investigators to probe for receptor-mediated control of net Cl transport since corneal epithelial health is dependent on innervation of this tissue. This dependence suggests that neurotransmitter release from nerve endings mediate – through receptor stimulation – responses supportive of corneal epithelial functions. Such a notion was validated by showing that loss of neural input results in impaired corneal epithelial regeneration and recurrent erosion. Accordingly, it has also been shown that the corneal epithelium expresses receptor subtypes responsive to adrenergic and cholinergic agonists released from sympathetic and parasympathetic branches of the autonomic nervous system. Other studies indicated that stimulation of these receptors elicit regulation of corneal epithelial renewal, ion transport activity, transparency, and fluid transport.

The regulation of cell volume by the aforementioned ionic transport mechanisms underlies the ability of the corneal epithelium to elicit net fluid transport from the stroma into the tears. It is possible that fluid transport is dependent on serial repetitive swelling and shrinking of cell volume. In this way, swelling occurs as a result of initial stimulation of net osmolyte uptake. Such rises occur in response to increases in Na+:K+ pump activity leading to rises in sodium chloride (NaCl) and potassium chloride (KCl) uptake through the Na+:K+:2Cl cotransporter. As a consequence of an increase in osmolyte uptake, fluid flow into the cells increases. Subsequently, the epithelial cell volume decreases as a result of stimulation of ionic efflux (i.e., KCl) into the tears. The swelling response is referred to as regulatory volume increase (RVI) due to increases in NaCl and KCl uptake. Subsequent shrinkage occurs as a consequence of RVI and is dependent on increases in KCl efflux leading to osmotically coupled fluid loss. This cell shrinkage is known as regulatory volume decrease (RVD). Therefore, fluid transport is tightly regulated by alternating stimulation and inhibition of osmolyte uptake and efflux into the corneal epithelium. How such tight and time-dependent control of these different transport mechanisms is mediated is unknown. It may be dependent on the ability of different receptor types to elicit – through a variety of second messenger pathways – rapid changes in the activities of these ion transporters mediating alternating RVI and RVD responses.

In addition to mediating fluid transport under isotonic conditions as a possible result of differential activation and inhibition of ion-transport activity and membrane permeability, RVD and RVI responses can be induced through variations in bathing solution osmolarity similar to those encountered in daily living and those identified in some types of dry eye disease. In order for cell volume regulatory responses to change fluid transport rates and thereby maintain corneal homeostasis, despite being exposed to an environmental hypertonic or hypotonic challenge, it is also necessary to have a coordinated regulation of ion transporters and parallel cell membrane permeability. Such regulation occurs through receptormediated events that can modulate ion transporter rates to either increase or decrease net ionic influx or efflux from corneal epithelial cells. These receptors elicit control of ion transporter function and cell permeability through second messengers that either concomitantly increase or inhibit ion transport rates through stimulation or inhibition of active ion transporter, cotransporters, and cell membrane permeability.

Such coordinated control is required for the maintenance of corneal transparency since they underlie RVD and RVI responses driving fluid out of stroma into the tears. Depending on the type of osmotic challenge, these responses sustain corneal epithelial barrier function. However, in individuals afflicted with dry eye disease, one problem is compromise of barrier function leading to stromal infection. Even though, in these patients, there may be activation of a RVI response due to chronic exposure to hypertonic tears, it may not be adequate to restore isotonic cell volume resulting in disruption of barrier function. This suggestion has prompted a host of studies over the last 30 years that are focused on characterizing receptor-mediated regulation of corneal epithelial active ion transport underlying regulatory volume responses. Their results have pointed investigators in directions that may lead to the identification of novel strategies for improving the rates of corneal epithelial renewal as well as restoring its transparency and refractive properties following an injury.

Other Transport Mechanisms

The limited corneal epithelial layer permeability presents a formidable barrier to solute and drug permeation into the ocular interior. This hindrance has prompted efforts to probe for the expression of transporters that could facilitate their uptake into the eye. A number of different transporters were identified that can elicit this effect. They include a carnitine/organic cation transporter and a sodium-dependent amino acid transporter. On the other hand, rather than identifying a drug-influx mechanism,

a multi-drug-resistant (MDR)-1 efflux mechanism has been described. Such an efflux process does not promote drug uptake into the ocular interior if they are substrates for the MDR-1 pumps. Therefore, improving drug delivery into the ocular interior is in most cases focused on increasing their hydrophobicity so that they can more readily permeate by membrane diffusion. One example of such an improvement is exemplified by rendering epinephrine more lipid soluble through its derivatization with a lipid. An alternative is to inhibit MDR-1-elicited drug efflux. However, none of the currently used inhibitors have adequate selectivity for this purpose.

Receptor-Mediated Control of Corneal Epithelial

Ionic Transport Functions

Adrenergic subtype (a1, a2, and b), serotonergic, and cholinergic receptor-linked functions have been identified in the corneal epithelium. Adrenergic receptors mediate their control of ionic transport and membrane permselectivity through second messenger pathways involving increases in intracellular Ca2+ and modulation of adenylate cyclase activity. Such variations change either the basolateral or apical membrane permeabilities resulting in modulation of active Cl transport through changes in the intracellular potential difference. For example, b-adrenergic receptor stimulation has a corresponding effect on net Cl transport by enhancing K+ basolateral membrane permselectivity as well as increasing Cl electrodiffusion across the apical membrane into the tears. Less is known about the signaling pathways eliciting cholinergic and serotonergic receptor control of net ion transport.

The corneal epithelium and accessory anterior ocular tissues express a host of cytokines that are critical to the maintenance of corneal epithelial functions. In particular, cytokine expression is critical for inducing control of cell proliferation, migration, and terminal differentiation. Their importance has become self-evident from studies on the mechanisms of corneal epithelial wound healing induced by injury. Numerous cytokine expression levels are upregulated to hasten corneal epithelial wound healing through stimulation of cell migration and proliferation. Such a realization has heightened the interest in determining the mechanisms of regulation of cell-signaling pathways linking their cognate receptor activation to these responses. It is believed that these studies will identify potential drug targets to improve in a clinical setting the outcome of injury-induced corneal wound healing.

Active ion transporters and membrane channels underlying receptor activation are components of a myriad of cell signaling pathways that mediate cytokine receptor control of epithelial renewal. One of the most potent and efficacious mitogens hastening corneal epithelial renewal is epidermal growth factor (EGF). This mitogen induces increases in cell proliferation and migration through

stimulation of different ion-transport mechanisms, ionic permeabilities, and receptor-linked channels. They include increases in the activity of the basolateral membrane localized Na+:K+:2Cl cotransporter resulting from stimulation of plasma membrane Ca2+ influx and activation of the mitogen-activated protein kinase (MAPK) superfamily. The increase in Ca2+ influx through a Ca2+ channel is dependent on EGF receptor (EGFR)-induced stimulation of membrane-associated phospholipase C (PLC) followed by hydrolysis of a phosphoinositide to induce Ca2+ release from an intracellular Ca2+ store. Another ionic influx affected by EGFR stimulation is K+ efflux through the basolateral membrane. In addition, mitogenic responses to EGF require that during cell-cycle progression cell volume is modulated to accommodate increases in the parent cell of genomic content prior to cell division. Such modulation is, in part, dependent on changes in K+:Cl cotransporter expression and activity. Another example of a cytokine whose induced effects are dependent on modulation of channel activity is the tumor necrosis factor-alpha (TNFa).

Cell-Volume Control and Epithelial Renewal

Epithelial renewal is a dynamic process that is dependent on tight regulation of cell volume. Prior to cell division, volume expansion of a parent cell is required to accommodate doubling of the nuclear and cytoplasmic components in preparation for their equal distribution into daughter cells. Similarly, changes in cell volume are requisite for cell migration, as this process involves repeated and coordinated leading-edge cytoplasmic volume extension, along with retraction at the opposite pole. As modulation of iontransport activity and membrane permeability underlie changes in cell volume, cytokine-induced control of renewal is, therefore, dependent upon modulation of ionic influx and efflux. Such control requires that there is synchronized release of specific cytokines that alter ion transport at appropriate times during the cell cycle. Numerous cytokines mediate the required regulation for error-free proliferation and migration. Therefore, cytokine receptor control of ion transport activity is of critical importance in the maintenance of corneal epithelial renewal, transparency, and its refractive properties.

Ca2+ Channel and Pump Activity

Receptor-induced Ca2+ signaling contributes to the regulation of net Cl transport and proliferation of corneal epithelial cells. In order for Ca2+ to link receptor activation to these responses, its intracellular concentration must be regulated at sub-micromolar levels.

Such regulation occurs through different types of Ca2+/Mg2+-ATPase plasma membrane and endoplasmic reticulum transporters and channels. The plasma membrane pump counterpart is selectively stimulated by