The pathogenesis of corneal epithelial defects

A C TA 0 P H T H A L M 0 L 0 G I C A 67 (1989) Supplementurn 192

The pathogenesis of corneal epithelial defects

Michael Berman

The Wilmer Eye Institute, The Johns Hopkins Hospital, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

Clinicians have long realized that persistent or recurrent epithelial defects, whatever the specific etiology, are correlated with subsequent stromal ulceration (Kenyon 1982). Until recently (Berman et al. 1983), however, the relationship between a defect and the mediators thought to contribute to stromal destruction has not been understood. Renewed interest in pathogenetic mechanisms has stimulated the study of adhesive relationships between epithelium and stroma, and of how those relationships are perturbed after corneal injury.

The normal human corneal epithelium (Fig. 1) is attached to the subjacent Bowman’s layer by a subepithelial basement membrane via hemidesmosomes, which are continuous structurally via anchoring filaments through the basal lamina and into the reticular lamina via anchoring fibrils. The epithelium is also thought possibly to be attached to basement membrane through the adhesive macromolecules fibronectin and laminin which mediate attachment of the basal epithelial cells to other integral components of the basal lamina, namely, heparan sulfate and type Iv collagen. Fibronectin (Berman et al. 1988), a dimer of approximately 440 000 daltons, contains distinct structural and functional domains which permit its binding to the cell surface and to other macromolecules of the basal lamina. The RGD (arginine-glycine-aspartic acid) recognition sequence (Ruoslahti & Piersch- bacher 1987) in the cell-binding domain of fibronectin is required for binding to integrin-type, fibronectin receptors at the cell surface. The other known adhesive glycoprotein in the basement membrane, laminin (Berman et al. 1988), molecu-

lar weight approximately one million daltons, is cross-shaped in appearance and, like fibronectin, has specific domains for binding to the cell surface and to heparan sulfate and type N collagen in the basement membrane. It is to be noted that to be effective fibronectin and laminin must be structurally continuous between epithelium and basal lamina (Berman et al. 1988). After simple injury to the cornea, for example, scrape-removal of epithelium, successful epithelial re-surfacing is thought to require epithelial attachment to and migration on (Fig. 1) a temporary copolymer of fibridplasma fibronectin. In this copolymer the fibronectin must be structurally intact initially such that the cell- binding domain is continuous with the matrix, that is, fibrin-binding domains, in order for attachment to matrix and epithelial migration to occur. In this model (Figs. 1,4) urokinase-like plasminogen activator (@A) is hypothesized to have an essential role in the normal, cyclic mechanisms of epithelial sheet attachment at the leading edge, migration and detachment; that is, epithelial resurfacing. Thus uPA, present in association with focal contacts at the basal cell surfaces of the cells at the leading edge of the epithelial sheet (Figs. 1,4) is activated in situ on the leading epithelial cell surface; and then the active uPA activates plasminogen to plasmin. Plasmin, in turn, cleaves through fibronectin in a limited fashion to release the leading edge of epithelium which then advan- ces to the adjacent fibronectin, in the haptotactic sense, to initiate another cycle. As in other systems, fibronectin is thought to initiate formation of focal contacts, perhaps by mechanisms involving ‘patch-

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56 Fig. 1.

- F-actin microfilaments to result in epithelial migration in the direction of the leading edge of adherent epithelium. After complete epithelial resurfacing, resorption of the remaining subepithelial fibridfibronectin is thought to be effected by plasmin; and the epithelium re-attaches to basement membrane and stroma via newly-made hemidesmosomes and associated anchoring structures. After an alkali bum, regulation of the system is thought, for yet unknown reasons, to go awry such that (Figs. 2,4) unregulated PA activity at the leading edge results in weaker and/or fewer attachments of epithelium to the subjacent fibrin/fibronectin to produce a secondary epithelial defect.

Fig. 1. Normal corneal re-.epitheliahation. a. Basal cells of the human corneal epithelium are attached to Bowman's Layer via a hemidesmosomalhasement membrane complex, in which anchoring filaments insert into the basal lamina and are continuous structurally via anchoring fibrils into the reticular laminia to end in an anastomosing system of anchoring plaques. The adhesive glycoproteins fibronectin (Fn) and laminin are also present in the basement membrane and are thought possibly to mediate attachment of basal epithelial cells to the basement membrane. b. After scrape removal of rabbit corneal epithelium, focal contact adhesive structures form at the leading edge of the migrating epithelium, in association with fibronectin receptor (integrin) and organized F-actin microfilaments, in response to matrix Fn (see below). c. After injury to the rabbit cornea (scrape-removal of epithelium or alkali burn), a copolymer of fibridfibronectin (fibronectin shown here by immunofluorescence one day post scrape) is formed on the stromal surface. Epithelial attachment to and migration on this copolymer is necessary for epithelial resurfacing of stroma. (arrow, leading edge of migrating epithelium). d. In the model of normal epithelial resurfacing, urokinase-like plasminogen activator (uPA) is hypothesized to have an essential role in the normal, cyclic mechanisms of epithelial sheet attachment at the leading edge, migration and detachment during resurfacing. Plasmin, generated by the uPA at the basal cell surface (perhaps in the focal contact zone) cleaves through the fibronectin in a limited fashion to release the leading edge of epithelium which then advan- ces in the haptotactic sence to adjacent fibronectin to initiate another cycle of 'make-slide-break' (see Fig. 4). After complete epithelial resurfacing, resorption of the remaining subepithelial fibrin/fibronectin is thought to be affected by plasmin; and the epithelium reattaches to basement membrane via newly-made hemidesmosomes and associated anchoring structures.

(1987) that the diuretic Amiloride inhibits urokinase-like activators (uPA) but not tPA, we have tested the ability of Amiloride to inhibit PA activity in sections of scraped or alkali-burned rabbit corneas. A.miloride was found to inhibit PA activity at the leading edge of scraped or burned corneas be- fore the stage of secondary epithelial defect formation. In burned corneas with secondary defects (Fig. 3) PA activity is present symmetrical to the epithelium, all across the cornea; and antibodies to tPA prevent lysis except at the leading edge. Ami- loride alone prevents lysis at the leading edge but not outside the epithelium more peripherally. The combination of Amiloride and anti-tPA com- pletely inhibits lysis in the sections. Amiloride (Fig. 3) also inhibits PA activity which begins at the leading edge of migrating rabbit and human corneal epithelial sheets in vitro. Thus, PA activity at the leading edge of corneal epithelium in vivo post scrape or burn and in vitro in tissue cultures of primary explants is thought to be due to uPA. In addition, Amiloride has been found to inhibit 46 and 35 kD uPA species obtained from cultures of ulcer- ating rabbit corneas with secondary epithelial defects. The Ki for inhibition of the high molecular weight uPA species is about 10 p M (Vassalli & Belin 1987; Berman 1988, unpublished data). Although antibodies to human urokinase have been found also to inhibit the rabbit uPA species in solution, such antibodies do not inhibit PA activity at the leading edge of migrating corneal epithelium, in either frozen sections of burned corneas with secondary defects or in tissue culture (Berman et al. 1989). It is thought that the antibodies, like extrin- sic protease inhibitors in some other systems (Blasi et al. 1987; Dan0 et al. 1985), do not have access to the cell-surface uPA, which is possibly integral in focal contacts. Hence, in this sense, the uPA at the leading epithelial edge might not be restrained by normal regulators, for example, the PAI-1 inhibi- tor of plasminogen activator. In the case also of persistent human corneal epithelial defects, as in a failed graft for bullous keratopathy (Berman et al. 1988) (Fig. 2), antibodies to tPA do not inhibit the leading edge activity; and uPA is thought to be present on the leading epithelial edge.

It is a working hypothesis (Fig. 4) that, after an alkali burn, fragments of fibronectin containing the cell-binding domain, separated by plasmin cleavage from matrix-binding domains, compete

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with intact fibronectin for the cell-surface fibronectin receptor; and matrix-binding domains of fibronectin compete with intact fibronectin for heparan sulfate in basement membrane and also for fibrin to prevent epithelial cell attachment and sheet migration; or cause detachment of epithelial cells from intact fibronectin in a subepithelial matrix to contribute to the formation of an epithelial defect. In the model presented (Figs. 2, 4-6), unregulated uPA generated at the leading edge of epithelium post burn generates plasmin that also eventually degrades the laminin component; and type IV collagenase, secreted by epithelium and activated by plasmin is thought to degrade the type IV collagen component of the subepithelial basement membrane. The shearing forces of lid ac- tion then act to pull away the loosely-adherent epithelium. In this view (Fig. 6) an epithelial defect, initiated by an unregulated cell-surface urokinase- like plasminogen activator is at the top of a cascade of cell-biologic and biochemical interactions which result in eventual stromal ulceration. In sup- port of this model, recent collaborative work by Ni- shida et al. (1988) has demonstrated that a peptide containing the RGD recognition sequence (Ruos- lahti & Pierschbacher 1987) of the cell-binding domain of fibronectin (GRGDS, glycine-arginine-glycine-aspartic acid-serine) can inhibit the attachment and spreading of rabbit corneal epithelial cells on fibronectin-coated culture dishes. Subse- quent work by Watanabe & Berman (1988) has demonstrated that GRGDS inhibits both chemo-

Fig. 2. Urokinase-like activator (uPA) and persistent defects. a. After an alkali bum, regulation of epithelial migration is thought to go awry such that unregulated urokinase-like PA activity (uPA) at the leading edge results in weaker and/or fewer attachments of epithelium to the subjacent fibrinifibronectin to produce a secondary epithelial defect. b. Continued generation of plasmin by unregulated uPA at the leading edge results in the degradation of basement membrane components and in the secretion and activation of type I (interstitial) collagenase in the corneal stroma to result in stromal ulceration. c. In a failed graft for bullous keratopathy (l), antibodies to tPA (a-tPA) inhibit PA activity in the actual defect region (4) but not that associated with the actual leading edge of epithelium, adjacent to the defect. This observation sug- gests that, as in the rabbit alkali burn model, uPA present at the leading epithelial edge in human material also has a role in the generation of persistent defects.

taxis and haptotaxis of rabbit corneal epithelial cells in response to plasma fibronectin gradients in the Boyden chamber (Fig. 4). Moreover, when GRGDS was added to cultures of rabbit corneas from which epithelium and basement membrane had been removed in vivo by scraping, the peptide retarded the rate of epithelial re-surfacing (Ber- man et al. 1988). The results are interpreted to mean that the RGD region, as in other fibronectin- dependent systems (Ruoslahti & Pierschbacher 1987), is necessary for mediating attachment of rabbit corneal epithelium to fibronectin; and that ‘un-coupled domains’ (Fig. 4) of fibronectin, generated by plasmin, might prevent both attachment to plasma fibronectin and migration of corneal epithelium in response to a haptotactic gradient of intact fibronectin, as presumed to be present under the leading edge of corneal epithelium (Figs. 1,4). Based on studies of epithelial defect formation (Berman et al. 1988; Hayashi et al. 1987) and stro- ma1 ulceration (Berman 1980) (Fig. 6), it is possible to resolve levels of interactions that are subject to clinical intervention. As in any cascade, however, the best potential for successful intervention lies at the top of the cascade, hence the emphasis clinically on re-establishing epithelial integrity. Trans- plantation of unburned conjunctival epithelium, championed by Thoft (1987), and Kenyon (1982), is one way to produce a stable epithelial surface; but the visual prognosis can be poor without subsequent penetrating keratoplasty. Current therapies (as in the treatment of non-healing human skin ulcers) also include the topical application of plasma fibronectin, as introduced by Dr. Nishida et al. (1983), to provide a temporary substratum for epithelial adhesion and migration. Since, it is known from the work of Salonen et al. (1987) that, regard- less of defect etiology, tears of patients with persistent epithelial defects contain high levels of active plasmin, aprotinin (Trasylol) is applied topically to inhibit plasmin at the corneal surface when en- dogenous or topically administered fibronectin is degraded inappropriately. Eventhough there have been improvements in non-surgical treatment mo- dalities, however, severely-injured corneas, as after alkali burns (Kenyon 1982), and corneas in patients with chronic systemic diseases, for example, rheu- matoid disease (Kenyon 1982), can develop epithelial defects and ulcerate despite all clinical attempts at management.

In previous work, epithelial wound healing has

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Fig. 3. Five-day post burn rabbit cornea with secondary epithelial defect: a. In the presence of control (non-immune) goat IgG, lysis is observed symmetrical to the peripheral epithelium (arrowheads, tPA), at the edge of epithelium adjacent to the defect (arrow, uPA), and in the defect region. b. Antibodies to tPA (500 pg/ml) inhibit the peripheral lysis associated with epithelium (arrowheads) but not that associated with edges of epithelium, adjacent to the defect (arrow). c. Ami- loride alone (1mM) inhibits lysis at the leading edge of epithelium (arrow) but not that symmetrical to the more peripheral epithelium (arrowheads) or in the defect region. d. The combination of Amiloride (1mM) plus antibodies to tPA (500 pg/ml) totally inhibits lysis associated with the section. Amiloride (1mM) inhibits uPA at the leading edges (arrows) of migrating rabbit and human epithelium in vitro: e. Lysis (arrow) begins, segmentally, in association with regions of outgrowth in primary cultures of rabbit corneal epithelium. f. In the presence of Amiloride, lytic activity is markedly reduced. PA (uPA) activity is clearly limited to edges of the migrating rabbit epithelium. g. Lysis (arrow) in primary cultures of human corneal epithelium also begins in association with edges of the explant (here lysis has continued be- yond the initial stage to obscure the original location of PA @PA) activity). h. In the presence of Amiloride, uPA-dependent lysis is observed to be much-reduced in magnitude and to be limited (arrow) to the edges of the human epithe-

lial explant.

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EPITHELIAL MIGRATION CYCLE Fig. 4. The pathogenesis of epithelial defects. a. Un- regulated PA activity (uPA) at the leading edge of

0 0 0 epithelium post bum is thought to generate plasmin that cleaves fibronectin to uncouple the cell-

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been studied in an ‘effective’ wound healing moL- (epithelium scrape-removed) in comparison to an ‘ineffective’ model (alkali-burned) in attempts to understand why secondary or persistent epithelial defects and stromal ulcers develop after alkali burns (Hayashi et al. 1987). The results of this study were interpreted to mean that tPA only was associated with epithelium during epithelial migration in the scrape model; whereas uPA activity was thought to appear at the leading edge of epithelium post burn, in association with the formation of secondary epithelial defects. More recent work

binding and matrix-binding domains. The uncoupled domains produced in high concentxa- tion subjacent to the leading epithelial edge are thought possibly to compete with intact fibronectin to generate a persistent epithelial defect. (scuPA, single chain UP& tcuPA, two chain uPA).

PATHOGENESIS OF EPITHELIAL DEFECT

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however (Berman et al. 1989) has demonstrated that uPA is also present on the leading edge of migrating epithelium post scrape, as on migrating skin keratinecytes (Monoka et al. 1987) and vascu- lar endothelial cells (Pepper et al. 1987). Thus, it now appears that the continued, abnormally-pro- longed presence of active uPA on the leading edge of corneal epithelium post alkali burn results in over-generation of plasmin, the accelerated degradation of subepithelial fibrinlfibronectin and a persistent (recurrent) epithelial defect. Indeed, the current renewed interest in the role of PA in tumor

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a

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Fig. 5. Model relating the leading edge of epithelium post burn to invasive metastatic tumor cells. Normal re-epithelialization is thought to involve cycling between stages in a and b. After an alkali burn, however, epithelium is thought, for yet un; known reasons, to be ‘trapped’ in stage b - resulting in cessation of further epithelialization and in the inappropriate excessive generation of plasmin and formation of ‘uncoupled domains’ of Fn containing the cell-binding domain which compete with intact Fn to prevent haptotactic interaction with Fn in the provisional matrix of FibrinIFn. Pos- sibly after an alkali burn, regulation of the scission of the N terminal region of uPA containing the EGF-like growth factor-like domain required for binding to uPA receptor goes awry such that uPA remains board to the cell surface (i.e. does not ‘cycle’) to result in sustained uPA activity in that location. Leading cells of a migrating corneal epithelial sheet post burn share common regulatory features with metastatic tumor cells, chiefly those concerning cell-extracellular matrix adhesion, cell migration and the roles of cell-surface uPA in those events. In addition to an hypothesized uPA (1igand)-receptor regulation of adhesion to fibronectin via fibronectin receptor (integrin), and the regulation of F-actin contractile mechanisms, of major concern are coupled stimulus-response transduction mechanisms, Ca++ flux regulation, Na+/H+ antiporter function and the regulation of F-actin assembly and disassembly in the lamellipodium, events presumed to be of importance in both the corneal epithelium and metastatic cell. The model that is proposed has the merit that it integrates regulatory features determined in other biological systems in order to permit tests of the possibility that uPA, bound to the cell surface via its EGF-like domain, and possibly in the focal contact zone, exerts ‘growth factor-like’ effects on the cell while located in a cell region that modulates both adhesion to fibronectin and proteolysis

at the leading edge of the cell.

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Fig 6. The cell hiology and biochemistry of epithelial defect formation and stromal ulceration. Based on studies of epithelial defect formation and stromal ulceation, it is possible to resolve levels of interactions that are subject to clinical intervention. As in any cascade, however, the best potential for successful intervention lies at the top of the cascade, hence

the emphasis clinically on re-establishing epithelial integrity.

invasion and metastasis (Blasi et al. 1987; Dan0 et al. 1985), is due to the specific correlation between the presence and levels of cell-surface urokinase- like PA and metastatis and to the reports that pharmacologic regulation of uPA synthesis and antibodies to uPA can suppress metastatis in ex- perimental models. In both corneal defect formation/stromal ulceration and tumor invasionlmeta- stasis, regulation of cell-extracellular matrix relationships is perturbed, leading to abnormal degradation of matrix components and to impaired function.

Our working assumption is that it is possible to modulate PA activity at the leading edge of migrating corneal epithelium to promote epithelial healing. In this regard, the cornea may also serve as a model for studies of tumor invasiveness and metastasis. In addition, studies of corneal wound healing might well yield insights into the mechanisms and treatment of chronic skin ulcers, where inap- propirate degradation of matrix fibronectin by the PA/plasmin system is thought to be pathogenetic (Frederick Grinnell, personal communication). The basic premise (Fig. 5) is that the leading cells of a migrating corneal epithelial sheet post burn share common regulatory features with metastatic

tumor cells, chiefly those concerning cell-extracellular matrix adhesion, cell migration and the roles of cell-surface uPA in those events. In addition to an hypothesized uPA (@and)-receptor regulation of adhesion to Fn via Fn receptor (integrin), and the regulation of F-actin contractile mechanisms, of major concern (Ruddon 1987; Pouyssegur 1987), are coupled stimulus-response transduction mechanisms, Ca" flux regulation, Na+/H+ antiporter function and the regulation of F-actin assembly and disassembly in the lamellipodium, events presumed to be of importance in both the corneal epithelium and metastatic cell. The model that is proposed has the merit that it integrates regulatory features determined in other biological systems (Blasi et al. 1987; Dana et al. 1985; Pouyssegur 1987), in order to permit tests of the possibility that uPA, bound to the cell surface via its EGF-like domain (Blasi et al. 1987), and possibly in the focal contact zone, as reported by Pollanen et al. (1988) for other cell types, exerts 'growth factor-like' effects on the cell while located in a cell region that modulates both adhesion to fibronectin and proteolysis at the leading edge of the cell. Thus, it is of great importance to understand the basis for the sustained occurrence of urokinase-like activator at

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the leading edge post burn as opposed to the switch from uPA back to tPA after closure of a defect post scrape, a situation in which epithelial resurfacing occurs without problem. Pharmacologic regulation of PA at the leading epithelial edge might then promote the healing of persistent epithelial defects and prevent corneal ulceration.

Acknowledgrnenst

This paper is dedicated to Claes-Henrik Dohlman, MD, who made the author aware of the clinical importance of persistent epithelial defects as well as of the contribu- tions one person can make to many others by setting an example for them of kindness, humility and honesty.

Figs. 1, 2, 4 and 6 are adapted with permission from Figs. in Berman et al. (1988): The Pathogeneses of Epil- thelial Defects and Stromal Ulceration. In: Cavanagh D (ed). The Cornea: Trans World Congres Cornea 111, pp 36, 38,39,40,41,42. Ravan Press, New York.

References

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Berman M, Manseau E, Law M & Aiken D (1983): Ulcera- tion is correlated with degradation of fibrin and fibronectin at the corneal surface. Invest Ophthalmol Vis Sci 2 4 1358-1366.

Berman M, Kenyon K, Hayashi K et al. (1988): The Pa- thogenesis of Epithelial Defect Formation and Stromal Ulceration. In: D Cavanagh (ed). The Cornea: Trans World Congr Cornea III, p 35: Raven Press, New York.

Berman M, Hayashi K, Young E, Pease S, Smith D, El- Ghatita, Askew M & Cragoe Jr J (1989): Urokinase-like plasminogen activator, corneal epithelial-migration and defect formation. Invest Ophthalmol Vis Sci (Abstr) in press.

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Morioka S, Lazarus G. Baird J & Jensen P (1987): Migrat- ing keratinucytes express urokinase-type plasminogen activator. J Invest Dermatol88: 418-423.

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Pouyssegur J (1987): The Growth Factor-Activatable Naf/H+ exchange system: a genetic approach. In: Bradshaw R & Prentis S (eds). Oncogenes and Growth Factors, p 292. Elsevier, New York.

Pollanen J, Hedman K, Nielsen L et al. (1988): Ultrastruc- tural localization of plasma membrane-associated urokinase-type plasminogen activator at focal contacts. J Cell Biol 106: 87-95.

Ruddon R (1987): Cancer Biology, second edition, p 324. Oxford, New York.

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Salonen E-M, Tervo T, Torma E et al. (1987): Plasmin in tear fluid of patients with corneal ulcers: basis for a new therapy. Acta Ophthalmol (Copenh) 65: 3-12.

Thoft R (1987): Conjunctival surgery for corneal disease. In: G Smolin & R Thoft (eds). The Cornea, second edition, p 577. Little, Brown, Boston.

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Author's address:

The Wilmer Eye Institute, The Johns Hopkins Hospital, 600 N. Wolfe St., Baltimore, MD, 21205, USA.

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The pathogenesis of corneal epithelial defects