A Tissue Culture Assay of Corneal Epithelial Wound Closure

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1 A Tissue Culture Assay of Corneal Epithelial Wound Closure Marcia M. Jumblarr and Arthur H. Neufeld Experimental assays have been developed using cultured tissue derived from rabbit corneal epithelium to study migration of epithelial sheets during wound closure and cell-substrate adhesion. To study wound closure, epithelial defects, 6 mm in diameter, were produced in vitro in 24 well multiplates by a local freezing technique, and the size of the remaining defect was quantitated over time by staining. To study adhesion, cultured cells were labeled with 3 H-leucine, suspended, and added to fresh culture plates. At various times, adherent cells were lysed and the radioactivity of the lysate was determined. Serum enhances the closure of experimental defects, but laminin and fibronectin have no effect. Agents which alter mitotic rate, such as epidermal growth factor and 5-fluorouracil, do not influence the rate of wound closure in this assay. Compounds which elevate intracellular levels of cyclic AMP inhibit wound closure but promote cell-substrate adhesion. Thus, cultured corneal epithelial cells may be used to assay for influences on the migratory events governing closure of superficial epithelial wounds. Invest Ophthalmol Vis Sci 27:8-13, 1986 The corneal epithelium closes superficial wounds by migration of the epithelial sheet over the denuded stroma. Mitosis ceases at the wound periphery, and a cohesive layer of epithelial cells slides toward the central wound area. l>2 During this time, cells adhere reversibly to the corneal surface in the absence of attachment organelles and sustained migration is dependent upon continued synthesis of cell surface glycoproteins. 3 ' 4 Once coverage of the wound is complete, migration ceases, and the epithelial monolayer re-forms stable hemidesmosomal attachments to its basement membrane. 3 Mitosis then resumes, resulting eventually in a mature stratified corneal epithelium. 1 Recently, we have established and characterized cultures of rabbit corneal epithelial cells. These cells require cholera toxin for optimal growth, form partially stratified cell layers with apical microvilli, and retain their ability to respond to /3-adrenergic agonists with increased cyclic AMP synthesis. 5 We have used these cell cultures to describe aspects of corneal epithelial wound healing, to study the effects of pharmacological agents on wound closure and cell-substrate adhesion, and to develop an assay which may prove useful as an alternative to in vivo toxicity testing. From the Ophthalmic Pharmacology Unit Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts. This work was supported by National Institutes of Health grant EY Submitted for publication: November 14, Reprint requests: Dr. Arthur H. Neufeld, Eye Research Institute, 20 Staniford Street, Boston, MA Materials and Methods Corneal Epithelial Cultures Rabbit corneal epithelial cell cultures were initiated from Dispase II treated corneas as previously described. 5 New Zealand white rabbits, used as a source of corneal tissue, were treated in accordance with the ARVO Resolution on the Use of Animals in Research. Cultures were established in the absence of a feeder layer in a medium consisting of equal parts of Dulbeccos Modified Eagles Medium and Hams F12 supplemented with 5% fetal bovine serum, cholera toxin (0.1 Mg/ml), epidermal growth factor (10 ng/ml), insulin (5 n%/m\), gentamicin (5 /ig/ml) and dimethylsulfoxide (0.5% v/v). Epithelial cells derived from six corneas were pooled and plated into the 6 wells of a 35-mm diameter multiplate. Cultures were fed three times weekly. After 7-10 days the confluent multilayer (cf reference 5 for morphology) were subcultured, and the cells derived from each 35-mm diameter well were plated into a 24-well multiplate, each well of which contained 1 ml of the above medium, except that cholera toxin was omitted. In some experiments, cells were plated into 6-well multiplates in the presence or absence of cholera toxin as indicated. Wound Closure Assay Discs, 6 mm in diameter, were cut from Millipore HA filters, rinsed in 6 changes of distilled water, boiled in distilled water, and dried in a laminar flow hood prior to use. A disc was placed on the surface of each

2 No. 1 CORNEAL EPITHELIAL WOUND CLOSURE / Jumblorr and Neufeld Fig. 1. Experimental wound closure assay: Corneal epithelial cells were subcultured into 24well multiplates and grown to confluency. Wounds were produced in quadruplicate by freezing at the indicated times before fixing and staining. Control (CON) were not wounded. Dark areas are remaining cellular areas; light areas are remaining cell-free wound areas. 24 Con hrs culture of a 24-well multiplate, gently tapped down, and a stainless steel probe (6 mm in diameter) was cooled in liquid nitrogen and placed against the plastic surface opposite the disc for 5 sec. The probe was then removed, medium consisting of Dulbeccos Modified Minimal Essential Medium and Hams F12 with the addition of gentamycin and dimethylsulfoxide and with or without other components as indicated was added, and the disc carefully lifted out leaving a discrete circular defect in the ceil layer. To determine the time course of wound closure, wounding was done in replicate fashion at staggered intervals, usually 0, 6, 24, 32 and 48 hr before the fixation step. At the time the earliest set of replicate control wounds would be almost closed, all cultures were drained of medium, fixed with neutral buffered formalin, and stained in situ with full strength Giemsa (see Fig. 1 for an example). The size of the remaining defect at the staggered time intervals, as revealed by the unstained cell-free area, was determined by projecting the plates onto a screen with an overhead projecter at a fixed distance, tracing the unstained area onto paper, and cutting out and weighing the remaining wound area. The weight was normalized, as a percentage, to that of the initial wound size or converted to area in mm 2 for comparison of wound sizes. To determine the effects of various agents on wound closure, all 24 wounds were made at 0 hr and fixed and stained at 42 hr. Relative wound size was determined as above. Mitotic Rate Cells derived from primary culture were plated in a 24-well multiplate at 1 X 104 cells/well. One ml of culture medium containing 5-fluorouracil (5-FU) and/ or epidermal growth factor (EGF) at the indicated concentration was added to each well. At the indicated times, cell number was determined by hemacytometer count. Cyclic AMP Synthesis Synthesis of cyclic AMP by cultured corneal epithelial cells was measured in cells subcultured in both the presence and absence of stimulators of adenylate cyclase. Medium was removed from the 6- or 24-well culture dishes and replaced with physiological buffer containing 0.5 mm isobutylmethylxanthine, a phosphodiesterase inhibitor.5 After 20 min, the buffer was removed and the adherent cell layer was lysed in 0.1 M KOH at 100 C. This alkaline extract was neutralized with an equal volume of 0.1 N HC1, and the resulting protein precipitate was removed by centrifugation (2000 X G, 15 min, 4 C), solubilized with 1 N NaOH and quantitated by the method of Lowry et al.6 Cyclic AMP concentration in the supernatant was determined by radioimmunoassay using commercially available reagents. Rates of cyclic AMP synthesis are expressed as the mean ± SEM (number of observations) of pmoles cyclic AMP/mg protein/20 min.

3 10 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1986 Vol. 27 T3 O Hours Fig. 2. Time course of wound closure in vitro: Wounded cultures were incubated in the presence ( ) or absence (O) of 5% fetal bovine serum and the size of the wounds determined and plotted as a function of time after healing. Mean ± SEM (n = 4). Cell-Substrate Adhesion Assay Corneal epithelial cells were grown to confluency and then labeled with 1 ^Ci/ml 3 H-leucine (300 mci/ mmol) for 18 hr. Cells were removed from the culture CM E CO CD 10-- dish with Dispase, centrifuged, resuspended in HEPES buffered Dulbeccos Modified Eagles Medium, triturated with a flame polished pipet, and passed through 2 layers of 120 u mesh nylon cloth. The resulting single cell suspension was adjusted to 1-5 X 10 4 cells/ml in the same medium at 4 C. Aliquots containing 1-5 X 10 3 cells were placed in the 24 wells of a multiplate, each well containing 1 ml of medium. Attachment proceeded for various times up to 30 min at 22 C in room atmosphere. At the end of the attachment period, medium and unattached cells were removed and the adherent cells were rinsed twice with 1 ml medium. Adherent cells were lysed with 1 ml 0.1 N NaOH containing 1% Triton X-100 and samples of the lysate, as well as aliquots of the original cell suspension, were placed in scintillation fluid to determine radioactivity. Radioactivity in the adherent cells was expressed as a percentage of that present in the initial cell aliquot. Materials, Drugs, and Supplies Tissue culture multiplates were obtained from Falcon (Oxnard, CA); medium and serum from Microbiological Associates (Walkersville, MD), Dispase II from Boehringer-Mannheim (Indianapolis, IN); insulin and epidermal growth factor from Collaborative Research (Waltham, MA); 5-fluorouracil, cholera toxin and forskolin from Calbiochem (San Diego, CA); 3 H- leucine and components of cyclic AMP radioimmunoassay from New England Nuclear (Boston, MA); type HAfiltersfrom Millipore (Bedford, MA); laminin and fibronectin from Bethesda Research Labs (Gaithersburg, MD) and isoproterenol, isobutylmethylxanthine, Giemsa stock solution, and Triton X-100 from Sigma (St. Louis, MO). Results Cyclic AMP Levels in Cultured Cells C 3 O % FCS Fig. 3. Effect of fetal calf serum (FCS) on wound closure. Cultures with experimental wounds were incubated in the presence of the indicated serum concentrations and at 42 hr were fixed and stained. Mean ± SEM (n = 6). Primary corneal epithelial cultures synthesize cyclic AMP at the rate of 22 ± 3 (6) pmol cyclic AMP/mg prot/20 min. The continued presence of cholera toxin in the primary tissue culture medium greatly enhances the rate of epithelial cyclic AMP synthesis to 386 ± 47 (6) pmol cyclic AMP/mg prot/20 min. However, cells originally grown in cholera toxin-containing medium and subcultured into cholera toxin-free medium for one wk have essentially basal rates of cyclic AMP synthesis 46 ± 8 (6) pmol cyclic AMP/mg prot/20 min. We have, therefore, used cholera toxin-free subcultures, derived from cholera toxin-containing primary cultures, to assay the effects of drugs on wound closure and cell-substrate adhesion.

4 No. 1 CORNEAL EPITHELIAL WOUND CLOSURE / Jumblorr and Neufeld 11 Wound Closure Model The wounds obtained by local freezing are discrete and of uniform size with an area of approximately 28 ± 0.2 mm 2 (mean ± SEM) for 24 wounds. The epithelial cells in the wound that are damaged adhere to the filter disc as it is lifted away, leaving a clearly demarcated cell-free wound area (Fig. 1). The remaining epithelial cells, moving as a cohesive sheet, gradually cover the easily visualized and quantitated central wound area. When wounds are made in replicate fashion and sequentially over time, a time curve can be constructed by relating the remaining wound area to the original wound area and the rate of closure determined (Fig. 2). When wounded cultures are incubated at 37 C in the presence of medium containing 5% fetal bovine serum, the central area is re-covered at a rate of 0.8 mm 2 /hr over the first 30 hr, and closure is generally complete before 48 hr (Fig. 2). Because a discrete and measurable wound remains after 42 hr of closure, the size of the wound at that interval was used as a measure to define the basal conditions for wound closure. In serum-free medium, epithelial wounds closed slowly; over 30% of the original wound remained at 42 hr (Fig. 2) and continued incubation for up to 72 hr did not lead to further closure. The effect of fetal bovine serum is dose dependent (Fig. 3) and cannot be mimicked by 5 or 20 mg of the serum attachment factor fibronectin or by 10 mg laminin (data not shown). Thus, serum is necessary for complete wound closure in this assay and is included at a concentration of 5% (v/v) in the medium in experiments designed to determine the effect of drugs on wound closure. Effect of Mitosis on Wound Closure Corneal epithelial mitotic rates were determined in cultures grown in the presence of an epithelial growth promoter, EGF, and an antimitotic agent, 5-FU. EGF at 10 /ig/ml enhanced the cellular mitotic rate; whereas, 5-FU at 10 Mg/ml totally suppressed EGF stimulated mitosis. Nevertheless, when added to cultures at the time of wounding, these agents had no effect on wound closure (Table 1). Effect of Cyclic AMP Synthesis on Wound Closure Corneal epithelial cultures were wounded and exposed to test agents for 42 hr, at which time cyclic AMP synthesis and wound closure were measured. Cholera toxin (0.1 Mg/ml) and forskolin (10~ 5 M) increased cyclic AMP synthesis and slowed wound closure as shown in Figure 4. Neither of these agents, however, completely prevented migration, and substantial closure occurred in all cases. Cultures treated Table 1. Effects of growth modulators on cell density and wound closure Modulator Control EGF EGF + 5FU Cell Number* (X10 4 ) 1.8 ± ± ±0.2 Wound Area\ (mm 2 ) 4.3 ± ± ± 0.7 * Determined in triplicate 7 days after innoculation of 1 X I0 4 cells. \ Determined in quadruplicate 42 hr after wounding. with the /3-subunit of cholera toxin (data not shown) or isoproterenol (10~ 5 M) had unchanged rates of cyclic AMP synthesis and wound closure. Cell Adhesion After 18 hr in medium containing 3 H-leucine, confluent cultures of epithelial cells had incorporated label at a rate that produced 1-3 counts/min/cell, providing a simple method for subsequent estimation of the number of adherent cells. Thirty minutes after addition of suspended cells to HEPES buffered medium at am I ^ > a 80 < o Q 20- CON ISOP CTX FS CON ISOP CTX FS Fig. 4. Effect of cyclic AMP synthesis on wound closure. Cultures were wounded and exposed for 42 hr to cholera toxin (CTX), isobutylmethylxanthine (IBMX), forskolin (FS), or isoproterenol (ISOP) added to medium containing 5% FCS. At this time, remaining wound area and cyclic AMP synthesis were determined. Mean ± SEM (n = 4).

5 12 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1986 Vol T Minutes Fig. 5. Effect of cholera toxin on cell substrate adhesion. Epithelial cell cultures grown in cholera toxin free medium were exposed to cholera toxin (1 Mg/ml) and labelled with 3 H-leucine for 18 hr. Cells were then suspended and added to replicate tissue culture wells. At the indicated times, medium and unattached cells were removed and the adherent cells lysed. Radioactivity of the lysate was determined as a percentage of the total radioactivity of the cell suspension. Cholera toxin treated ( ), control (O). Mean ± SEM (n = 6). bient temperature, 30-50% of cells derived from cholera toxin supplemented cultures had adhered to the plastic substrate; whereas, during the same time only 10-15% of the cells derived from cholera toxin-free cultures adhered (Fig. 5). When collagen coated wells were used as a substrate, 30% of cells grown in the absence of cholera toxin adhered in the 30 min-assay period. When cells grown in the presence of cholera toxin were exposed to the collagen substrate, there was no further increase in adhesion of these cells as compared to cells exposed to the plastic substrate. Discussion Superficial epithelial defects close by epiboly, migration of the epithelial cell sheet over the basal lamina. The corneal epithelium, which consists of stratified squamous cells and rests on an avascular matrix, is a particularly simple system in which to study epithelial migration. Corneal epithelial wound closure has been studied both in vivo and in organ culture.'' 2A7>8 In the present study, we have demonstrated that cultured cells of the corneal epithelium in culture retain the ability to re-cover a model defect. The rate of wound closure in vitro in the presence of serum, as determined by linear regression analysis of the rate curve in Figure 2, is 0.8 mm 2 /hr and is similar to that observed in experimental wounds in vivo. 7-9 For wound closure to occur in vitro, serum is a necessary component of the medium. Recently, a serum protein, epibolin, was isolated which, when added to epidermal organ cultures, promotes migration of the epithelial sheet. 10 Whole serum, added to corneal organ cultures, promotes migration of the epithelium, and the attachment factor fibronectin, a serum component, has been identified in the basement membrane zone of healing corneas." 12 However, our results demonstrate that the permissive factor for migration in serum is unlikely to be fibronectin or laminin. In vivo, wounds close initially by migration of epithelial cells from a peripheral site rather than proliferation within the wound site. The addition of EGF, which is mitogenic for the cultured epithelial cells, to the experimental medium has a negligible effect on closure. Conversely, the antimitotic agent, 5-FU, added in a concentration sufficient to suppress EGF-induced mitosis, has no effect on wound closure. We therefore conclude that migration, and not mitosis, is the dominant process by which wound closure occurs in this in vitro model. To cover superficial defects, migratory cells must continuously make and break cell-substrate adhesions. The "stickiness" of cells keeps them adherent to their substrate and prevents inadvertent damage to the healing tissue by mechanical trauma, such as the movement of the eyelid over the surface of the cornea during blinking. Only after migration ceases do epithelial cells re-form stable hemidesmosomal attachments to their basement membrane. 3 Cell substrate adhesion during migration is probably dependent upon glycoproteins on the cell surface. In the cornea, glycoproteins are normally synthesized by migrating cells, and tunicamycin prevents both glycoprotein synthesis and epithelial migration. 413 The present study shows that cholera toxin stimulated cyclic AMP synthesis increases the ability of cultured corneal epithelial cells to adhere to both plastic and collagenous substrates. Recent evidence demonstrates that dermal epithelial cells cultured in the presence of cholera toxin produce a specific adhesion protein, and that stimulated cyclic AMP synthesis increases the rate of secretion of epithelial adhesion glycoproteins Synthesis of the glycoprotein fibronectin by full thickness corneas is enhanced by cyclic AMP. 16 Thus, cholera toxin and forskolin, via cyclic AMP synthesis, may increase the secretion of adhesive cell glycoproteins, thereby increasing cell substrate adhesion. This may act to slow the rate of migration in culture as cyclic AMP, either added exogenously or synthesized endogenously, slows migration of several cell types. 17 The lack of effect of isoproterenol is probably due to the inability of this drug to sustain elevated cyclic AMP levels. The paradoxical effect of cholera toxin to stimulate wound closure in vivo may therefore be consistent with

6 No. 1 CORNEAL EPITHELIAL WOUND CLOSURE / Jumblorr ond Neufeld 13 its inhibitory effect in the culture model. Originally, we observed that pretreatment in vivo with topical cholera toxin produced an increased rate of closure of superficial corneal defects; whereas, we now report that, in vitro, cholera toxin slows closure. 56 We also observed earlier that cholera toxin slows wound closure in a full thickness corneal organ culture model in which the epithelium migrates over native substrate to close a superficial defect. 8 Thus the differences observed in vivo and in vitro are unlikely to reflect the sensitivity of epithelial cells to differing substrates. If cyclic AMP promotes adhesiveness of migrating cells in vivo, as it does in vitro, this mechanism will protect responding cells from mechanical disruption due to blinking and therefore appear to promote wound closure. In vitro, this mechanism will slow but not prevent migration. An alternative explanation for cholera toxin stimulation of wound closure in vivo may include enhanced epithelial proliferation. Cholera toxin does potentiate the mitogenic effects of insulin and EGF. 5 Cholera toxin may potentiate the influences of other cell types in vivo or mediators that are absent in epithelial tissue culture. Our data does not permit us to distinguish between these hypothetical mechanisms. Cultured corneal epithelial cells provide a useful model system for investigating the events governing closure of superficial epithelial defects and for assaying the actions of exogenous agents on these events. Tissue culture offers several advantages over in vivo and organ culture models of wound closure: (1) The extracellular milieu including nutrients, growth factors and substrate is easily manipulated; (2) Closure of defects occurs in the absence of other cell types, such as nerves, mesenchymal fibroblasts, or inflammatory cells; (3) A large number of cultures, in this case 24, can be derived from a single cornea; and (4) Cells can be easily labelled and used for cell-substrate adhesion determinations. Multiplate cultures of corneal epithelium should prove useful for investigating the influence of a variety of modulators, drugs and chemicals on wound closure under defined conditions. Establishment and routine assay of corneal epithelial wound closure in multiwell plates may also prove useful as an in vitro, toxicological test and an alternative to animal testing. Key words: cornea, epithelium, adenosine cyclic monophosphate, wound closure, migration, tissue culture Acknowledgments We thank Gail Raymond and Nancy McLaughlin for their excellent technical assistance. References 1. Hanna C: Proliferation and migration of epithelial cells: during corneal wound repair in the rabbit and the rat. Am J Ophthalmol 61:55, Kuwabara T, Perkins DG, and Cogan DG: Sliding of the epithelium in experimental corneal wounds. Invest Ophthalmol 15: 4, Buck RC: Hemidesmosomes of normal and regenerating mouse corneal epithelium. Virchows Arch (Cell Pathol) 41:1, Gipson IK, Kiorpes TC, and Brennan SJ: Epithelial sheet movement: effects of tunicamycin on migration and glycoprotein synthesis. Dev Biol 101:212, Jumblatt MM and Neufeld AH: /3-Adrenergic and Serotonergic responsiveness of rabbit corneal epithelial cells in culture. Invest Ophthalmol Vis Sci 24:1139, Lowry OH, Rosenbrough NJ, Farr AL, and Randall RJ: Protein measurement with the folin phenol reagent. J Biol Chem 193: 265, Jumblatt MM, Fogle JA, and Neufeld AH: Cholera toxin stimulates adenosine 3',5'-monophosphate synthesis and epithelial wound closure in the rabbit cornea. Invest Ophthalmol Vis Sci 19:1321, Jumblatt MM and Neufeld AH: Effects of cyclic AMP and calcium on corneal epithelial wound closure in vitro. ARVO Abstracts. Invest Ophthalmol Vis Sci 22(Suppl):25, Jumblatt MM and Neufeld AH: Characterization of cyclic AMPmediated wound closure of the rabbit corneal epithelium. Curr Eye Res 1:189, Stenn KS: Epibolin: a protein of human plasma that supports epithelial cell movement. Proc Natl Acad Sci (USA) 78:6907, Nishida T, Nakagawa S, Ohasi Y, Awata T, and Manabe R: Fibronectin in corneal wound healing: appearance in cultured rabbit cornea. Jpn J Ophthalmol 26:410, Fujikawa LS, Foster CS, Harris TJ, Lanigan JM, and Colvin RB: Fibronectin in healing rabbit corneal wounds. Lab Invest 45: 120, Gipson IK and Kiorpes TC: Epithelial sheet movement: protein and glycoprotein synthesis. Dev Biol 92:259, Okamoto Y, Sakai H, Sato J, and Akamatsu N: Effects of dibutyryl cyclic AMP on the synthesis of dolichol-linked saccharides and glycoproteins in cultured hepatoma cells. Biochem J 212: 859, Roberts GP and Jenner L: Glycoproteins and glycosaminoglycans synthesized by human keratinocytes. Biochem J 212:355, Nishida T, Tanaka H, Nakagawa S, Sasahe T, Awata T, and Manabe R: Fibronectin synthesis by the rabbit cornea: effects of mouse epidermal growth factor and cyclic AMP analogs. Jpn J Ophthalmol 28:196, Dunlap MK: Cyclic AMP levels in migrating and nonmigrating newt epidermal cells. J Cell Physiol 104:367, 1980.