JOURNAL OF CELLULAR PHYSIOLOGY 145:549-554 (1990)
Differential Modes of Action of Fibronectin and Epidermal Growth Factor on Rabbit Corneal Epithelial Migration TERUO NISHIDA,' MASATSUGU NAKAMURA, HlROSHl MISHIMA, AND TOSHlFUMl OTORI Department of Ophtha!mology, Kinki University School of Medicine, Osaka-Sayama City, Osaka 589, japan In order to clarify the roles of fibronectin (FN) and epidermal growth factor (EGF) in corneal wound healing, we cultured blocks of excised rabbit cornea for 24 hours in media containing one of these agents, then measured the length of the path of the epithelial layer that had migrated down the side of the block. Both FN and EGF stimulated epithelial migration significantly in a dose-dependentfashion. Responses to EGF involved a time lag of at least 12 hours before stimulation could be observed, but there was no lag-time for FN-stimulated migration. FN was maximally effective only if it was continuously present. In contrast, exposure to ECF for 6 hours did not stimulate epithelial migration, but exposure for 9 hours resulted in the same stimulatory effects as were observed after 24 hours' continuous exposure. Anti-FN antibody inhibited the FN- and EGF-stimulated migration of corneal epithelium. But anti-EGF antibody inhibited only EGFstimulated migration and had no effect on FN-stimulated migration. These results indicate that, unlike FN, EGF need not be present, once the epithelial cells have recognized its signal. Furthermore, the stimulatory effect of EGF depended on FN, while that of FN was independent of EGF. The effects of EGF on migration of corneal epithelium may, therefore, be mediated by FN.
The cornea consists of epithelium, stroma, and endothelium. The stroma has a regularly layered structure of collagen fibers and proteoglycans, such as keratan sulfate (Kenyon, 1987). An amorphous condensation of collagen forms the Bowman's membrane, on which the basement membrane of the epithelium rests. Basal cells rest on this basement membrane, whose extracellular matrix proteins, fibronectin (FN), laminin, and type IV collagen help to maintain the layered, striated structure of the corneal e ithelium. Maintenance of an intact corneal epithelia layer is an important defense a ainst infection, but the direct contact of the cornea wit the external environment often results in disturbance of the smooth corneal epithelium. The immediate resurfacing of orchestrated responses extracellular factors, cellular matrix roteins. Among the re orted factors that modulate t is migration of epitherium to cover defects, FN and epidermal growth factor (EGF) have been studied the most (Nishida, 1988; Tripathi et al., 1990).EGF binds to corneal epithelial cells (Fratiet al., 1972; Ho et al., 1974), stimulating wound closure (Savage and Cohen, 1973; Arturson, 1984; Petroutsos et al., 1984; h i m et al., 1988; Schultz et al., 1988a,b). The presence of EGF in tears was recently reported (Ohashi et al., 1989). FN and EGF both stimulate the migration of e ithelium in cultured rabbit cornea and facilitate the c osure of corneal epithelial defects in vivo (Frati et al., 1972; Savage and Cohen, 1973; Daniele
P
a
R
P
0 1990 WILEY-LISS, INC.
et al., 1979; Nishida et al., 1983a, 1984a; Arturson, 1984; Petroutsos et al., 1984; S igelman et al., 198.51, but their modes of action di fer (Ho et al., 1974; Watanabe et al., 1987). FN is chemotactic and haptotactic for corneal epithelial cells, but EGF is not (Watanabe et al., 1988). We previously reported that FN stimulated the attachment and spreading of cultured, single rabbit corneal epithelial cells on a FN matrix. Furthermore, FN stimulated migration of rabbit corneal e ithelium as a sheet in or an culture (Nishida et ay., 1983a) and in vivo (dshida et al., 1984a). The mechanism of the stimulatory effects of FN might be based on the adhesive characteristics of the FN molecule. Each domain of the FN molecule exhibits specific binding activity for collagen, fibrin, heparin, or its cellular receptor, known as inte in (Ruoslahti, 1981; Yamada, 1983; Hynes, 1985; Buc and Horwitz, 1987). FN is thought to bridge the cellular FN receptor and underlying extracellular matrix materials such as collagen. Indeed, the addition of GRGDS, the amino acid sequence of the cell-binding domain of FN, inhibited attachment and spreading demonstrating the presence of a FN receptor (integrin) in corneal epithelial cells (Nishida et al., 1988). Since the first report of Cohen (Cohen, 1962), the stimulatory effects of EGF have been studied extensively. Frati
F
iT
Received May 17, 1990; accepted September 6, 1990. 'To whom reprint requestskorrespondence should be addressed.
550
NISHIDA ET AL.
et al. (Frati et al., 1972) re orted that EGF rece tors are present in corneal epit elial cells and that GF stimulated DNA and RNA synthesis in corneal epithelial cells. Topical application of EGF was shown to stimulate reepithelization of various types of corneal epithelial defects (Daniele et al., 1979; Petroutsos et al., 1984; Singh and Foster, 1987; Schultz et al., 1988a,b). EGF stimulated the migration of corneal epithelium down the cut stromal surface of corneal blocks, apparently by stimulating cell proliferation, as measured by the incorporation of 3H-th midine (Watanabe et al., 1987). On the other hand, N does not stimulate DNA synthesis (Watanabe et al., 1987; Nishida et al., 1988). Diverse pathological conditions can lead to persistent epithelial defects, with or without ulceration of the corneal stroma. Use of FN and EGF for the treatment of such disorders is being investi ated. On a thin section taken from a block of culture cornea, epithelial migration can be visualized as a heavily stained cell layer extending art way down the side of the block, from what was t e anterior surface of the eye toward the endothelium. We measured the length of the path of the epithelium, which we refer to as the migration of the epithelium, on the blocks cultured with FN or EGF to determine whether the continuous presence of these agents is required for their maximum effect. We studied also the effects of antibodies against FN and EGF on this migration of epithelium. Information gained from our studies may be useful in optimizing the clinical application of these agents and in differentiating their use for different clinical sta es of persistent corneal epithelial defects, whose mec anisms of action are not completely understood. MATERIALS AND METHODS Albino rabbits wei hing 2 to 3 kg were obtained from Awazu Experimenta Animals (Settsu, Osaka, J a an), phosphate-buffered saline (PBS) and TC-199 cu ture medium were from the Research Foundation for Microbial Diseases of Osaka University (Suita, Osaka, Japan), and plastic multi-well culture dishes (24 wells, #3424) were from Costar (Cambrid e, MA). Human plasma FN was from the New York B ood Center (New York, NY). Recombinant human EGF and polyclonal anti-human EGF rabbit antiserum were from Earth Pharmaceutical (Akoh, H ogo, Japan). The purity of FN and EGF were assesse by SDS polyacrylamide gel electrophoresis and by high-performance liquid chromatography. Guinea pig antiserum against rabbit plasma FN was prepared as characterized and described previously (Nishida et al., 1983a). Polyacrylamide gel electrophoresis and Western blottin indicated that the antibodies used were specific or FN or EGF. Rabbit corneas were cultured as described previously, with sli ht modification (Nishida et al., 1983a; Watanabe et a f , 1987). Rabbits were sacrificed with an intravenous overdose of pentobarbital. Eyes were enucleated; a sclerocorneal section was excised, washed several times with sterile PBS, and cut into 2 x 4-mm blocks with a razor blade. The blocks were cultured in plastic multi-well tissue culture dishes with 1 ml of serum-free TC-199 culture medium with the experimental agents (human plasma FN, recombinant hu-
R
E
P
%
R
a
P
P
f
B
f?
man EGF, or antisera). Cultures were maintained at 37°C in a humidified incubator under 5%COz.The rate of epithelial mi ration was determined by examinin one series of cu tures after 6, 12, 18, or 24 hours. A1 oiher cultures were maintained for 24 hours; in the first of these experiments, the culture medium containing FN (100 kg/ml) was re laced by serum-free TC-199 medium after 6,12, or 18 ours; other specimens were cultured without FN for the first 12 hours and with FN for the last 12 hours. Another group of corneal blocks was incubated with FN for the entire 24 hours. The culture medium containing EGF (3 nglml) was replaced by serum-free TC-199 after 3, 6, 9, or 12 hours, and other groups of corneal blocks were cultured without EGF for the first 12 hours and with EGF for the last 12, or with EGF for the entire 24 hours. Control specimens were cultured in serum-free TC-199 medium alone. In the second series of experiments, s ecimens were cultured with various concentrations of N (1,10, and 100 p,g/ml) or EGF (0.3, 1,3, and 10 nglml) for 6 or 12 hours, and then the culture medium was replaced by serum-free TC-199 medium. Others were cultured at the same concentrations for the entire 24 hours. The third series of experiments employed the following agents: FN (100 kg/ml) with normal guinea pig serum, anti-FN antiserum (0.5,1.0,2.0,and 5.0 mg/ml) anti-FN anti-serum (at the same concentrations) with FN or with EGF (3 nglml) or normal uinea pig serum (control). Other cultures contained E F (3 ng/ml) with normal rabbit serum, anti-EGF anti-serum (1,3,10,30, or 100 Kgiml), anti-EGF antiserum (at the same concentrations) with EGF or FN (100 pg/rnl), or normal rabbit serum (control). Epithelial migration down the cut side of the block, toward the endothelial side, was measured as reported previously (Nishida et al., 1983a; Watanabe et al., 1987). In brief, the cultivated corneal blocks were washed with PBS and fixed overnight with a mixture of 100% ethanol and glacial acetic acid (955) at 4°C. The blocks were deh drated, immersed in xylene, and embedded in para in; three 4-km sections were cut, 300 bm apart, from each block, so that elon ation of the epithelial path could be determined. After deparaffinization, the specimens were stained with hematoxylin-eosin and observed under a light microsco e (Model BH-2, 01 mpus, Tok 0 , Japan). Photogra were taken with lack and w ite film (Neopan F, 8uji Films, Tokyo, J a an) with the aid of an automatic exposure meter. e length of the epithelial cell layer was measured on the photographs of each corneal block. Results were expressed as means ( * SEMI of 6 measurements. Statistical analysis was performed by Student's t-test. RESULTS Rates of migration of the epithelium are shown in Figure 1. The corneal epithelium cultured in serumfree TC-199 medium migrated as a sheet over the cut surface of the corneal stroma; the len h of the migrated epithelium did not increase signi icantly during the first 6 hours, but active migration was noted between hours 12 and 24 (P< .001). Epithelium cultured with FN (100 Fg/ml) also migrated slightly during the first 6 hours, and more rapidly thereafter,
I
K
K
i
8
H
B &
1s
l
8"
551
FN AND EGF ON CORNEAL EPITHELIAL MIGRATION
- 750
-
TABLE 1. Duration of exposure to F N or EGF related to corneal epithelial migration'
E
1
v
.-c 9 .-0
Exposure
500 -
period
FN (100pg/ml) W
(hr)
(rm)
None
-E
394+ 15 520+23* 4 3 0 f 27 431 *29
0-24 0-12 12-24
2ml 250 -
5 .n m
EGF (3ng/ml) Migration %I (wm) 389 f 23 100 579 f 44* 149 147 570 f 40*
Migration
100 132 109 109
377 f 30
97
* SEM (n = 6). * P < ,005 against controls. 'Means
00
6
12
18
24
hours in culture (hr)
Fig. 1. Chronological changes in the length of the path of corneal epithelium cultured in unsupplemented TC-199 (open circle), in TC-199 containing FN (100 pgiml, solid circle), and in TC-199 containing EGF (3 ngiml, open triangle). Bars show SEM of 6 measurements.
fF
but FN si ificantly increased the len h of the epithelial cel layer at 24 hours, compare with controls (P< ,001).In the presence of EGF (3 ng/ml), the length of 12 hours was increased only slightly compared with controls. After 24 hours, however, the length of the path was about 50% greater than that of specimens cultured in unsupplemented TC-199 medium (P < .001). These results also demonstrated two phases in the epithelial migration: a preparatory lag phase and an active migratory phase. The effect of the presence of FN (100 pg/ml) or EGF (3 nglml) during the first or the last 12 hours of the 24-hour incubation period is summarized in Table 1. When the corneal blocks were cultured in unsupplemented TC-199 culture medium, the length was 394 or 389 km. The continuous presence of FN or EGF increased the length of the path of 520 (P < .005) or 579 (P < .005) pm, respectively. Exposure to FN for the first or for the last 12 hours resulted in increases which were similar, and intermediate in magnitude between those seen when medium was unsupplemented or when FN was present for 24 hours. In contrast, exposure to EGF for the first 12 hours resulted in as much epithelial migration as that seen in specimens exposed to EGF continuously for 24 hours, whereas exposure to EGF for the last 12 hours did not stimulate the migration of epithelium at all. Thus, FN stimulated epithelial migration whether added early or late, whereas EGF produced an observable response only after it had been resent for a certain period of time. Because the thic ness of the cultured cornea was limited, we could not continue the cultivation beyond 24 hours. Thus it is difficult to conclude whether or not EGF would be effective if present during the late phase, when active migration was observed. The length of the migrated epithelium after 24 hours was proportional to the duration of exposure to FN (6, 12,18,or 24 hours) (Fig. 2). Response to EGF was more com licated; exposure to EGF for up to 6 hours did not resu t in any increase in epithelial migration, but exposure for 9 or 12 hours resulted in path lengths similar to those seen on corneal blocks cultured continuously with EGF for 24 hours (Fig. 2).
Y
R
P
* 14
0
500
250
** 150
epithelial migration (pm)
Fig. 2. Effects of the duration of exposure to FN (100 p iml, open column) or EGF (3 ng/ml, solid column) on the length of t i e path of corneal epithelium in corneal blocks cultured for 24 hours. Bars show SEM of 6 measurements. *P< .02 and **P< ,005against control.
When we tested whether these effects depended on the concentrations of FN and EGF, we found, in the case of FN, that the length of the path increased with the concentrations (1, 10, or 100 pg/ml), whether exposure had been for 6, 12, or 24 hours. As expected, the increases in the length of the epithelium after incubation with FN for 6 or 12 hours were less than those seen after incubation for 24 hours, even at the highest concentrations (Fig. 3). The length of the path of the corneal epithelium showed almost identical, concentration-dependent increases when blocks were cultured with various concentrations of EGF for 12 or 24 hours. However, no increase in len h was observed when the corneal blocks were culturec? for 6 hours with EGF, even at its highest concentration (10 ng/ml) (Fi . 4). hgure 5 shows the quantitative effects of FN and various concentrations of anti-FN antiserum. The addition of FN alone increased the corneal epithelial migration significant1 (P < .05). Anti-FN antiserum alone inhibited epithe ial migration significantly, in a dose-dependent manner, apparently abolishing the effects of endogenous FN at the higher concentrations (P< .001 at 5 mg/ml of anti-FN antiserum). The addition of anti-FN antiserum a t a concentration of 0.5 mg/ml completely reversed the stimulatory effect of
P
552
NISHIDA ET AL
750
1
E
a
I
1
0
10
100
0
concentration of FN (u g/ml)
r
1
I
I
3
10
concentration of EGF (ng/ml)
Fig. 3. Effects of the concentration of FN on the length of the path of corneal epithelium in corneal blocks exposed for 6 (0 en circle), 12 (solid circle), or 24 (open triangle) hours to FN. Bars siow SEM of 6 measurements.
750
0. 3
Fig. 4. Effects of the concentration of EGF on the length of the path of corneal epithelium in corneal blocks exposed for 6 (open circle), 12 (solid circle), or 24 (open triangle) hours to EGF. Bars show SEM of 6 measurements.
r
750 +,----~~---........_..__._.__.__~ ___.....___........ ..--.__..... -4
T
I
I
0
0. 5
1
5
2
0
0. 5
1
2
5
concentration of serum or antiserum (mg/ml)
concentration of serum or antiserum (mglml) Fig. 5. Effects of various concentrations of anti-FN antiserum and FN on the length of the path of epithelial migration after 24 hours. Open circle: anti-FN antiserum, solid circle: anti-FN antiserum and FN (100 pg/ml), open triangle: normal guinea pig serum, and solid triangle: normal guinea pig serum and FN (100 pg/ml). Bars show SEM of 6 measurements.
Fig. 6. Effects of various concentrations of anti-FN antiserum and EGF on epithelial migration. Open circle: anti-FN antiserum, solid circle: anti-FN antiserum and EGF (3 ngiml), open triangle: normal guinea pig serum, and solid triangle: normal guinea pig serum and EGF (3 ng/ml). Bars show SEM of 6 measurements.
exogenous FN (at 0.5 m /ml, P < .05, different from the group cultured with F alone; at 5.0 mg/ml, P < .001, different from the group cultured with FN alone). No statistically significant difference was observed between these specimens and those cultured with anti-FN antiserum alone. Non-immune guinea ig normal serum did not appear to influence epithefial migration. These results demonstrated that FN mediated migration of the corneal epithelium. Figure 6 shows the effects of various concentrations of anti-FN antiserum in the presence or absence of EGF. The presence of non-immune guinea pig normal serum did not affect e ithelial migration in the presence or absence of E F. The addition of EGF alone increased the length of the path of the corneal e ithelium significantly (P < .05), but the addition o antiFN antiserum (0.5 mg/ml) reversed this EGF-stimulated increase completely (P< .02). The addition of anti-FN antiserum inhibited epithelial migration in a dose-dependent fashion (at 5 mg/ml of anti-FN antiserum, P < .01 compared with specimens cultured with-
out EGF; P < ,001 compared with specimens cultured EGF). with __ Next we-investi ated the effects of various concentrations of anti-E F antiserum. The addition of antiEGF antiserum or rabbit normal serum affected neither epithelial migration nor the stimulatory effect of FN at any antiserum concentrations examined (data not shown). However, anti-EGF antiserum reversed the EGF-stimulated mi ration of the epithelium in a dosedependent fashion ( ig. 7).
If
8
P
~~~~
8
%
DISCUSSION Because a smooth, intact corneal epithelium is essential for the maintenance of good vision and defense of the cornea against infection, it is ver important to understand the mechanism(s1 that regu ate the resurfacing of epithelial defects. Recent advances in cell biology allow us to investigate the role of extracellular matrix and growth factors in corneal wound healing. The clinical efficacies of both FN and EGF for the treatment of non-healing corneal epithelial defects
T
553
FN AND EGF ON CORNEAL EPITHELIAL MIGRATION
750 r
grate. As we previously re orted, the addition of mouse EGF stimulated the synt esis and secretion of FN in cultured full-thickness rabbit cornea (Nishida et al., 1984b),indicating that EGF stimulates the synthesis of FN. FN secreted into the extracellular s ace may bind to cell surface receptors for FN, and t us stimulate epithelial migration, although it has not been clarified yet which kinds of corneal cells are responsible for FN synthesis. Recently, we found that the number of cells attached to the FN matrix increased when the corneal e ithelial cells were cultured with EGF, suggesting t at EGF might stimulate the expression of FN receptors by the corneal epithelial cells (unpublished data). Therefore EGF might act on the cornea in a combination of different ways: by increasing e ithelial cell numbers (proliferation), by increasin N synthesis # stimulating and secretion by the whole cornea, anc fby expression of FN receptors. The addition of anti-FN antiserum inhibited stimulation of epithelial migration by either FN or EGF. Furthermore anti-FN inhibited epithelial migration that would take lace without the addition of exogenous FN. The adfition of anti-EGF antiserum did not affect epithelial migration driven by FN, but did inhibit the stimulatory action of exo enous EGF. In the preliminary experiments, when t e higher concentrations of anti-FN was added, most of the epithelial cells detached from each other. Although anti-FN used in this study was not affinity purified, we noted that normal guinea pig serum did not affect epithelial migration and that anti-EGF. These findings suggest that the inhibition of normal e ithelial migration, in the absence of exo enous FN, is LTue solely to antiserum directed against N. These results demonstrate that the action of EGF on corneal epithelial cells might be mediated through FN. Recently Soong and his associates (1988a,b) reported that the addition of EGF or FN to the culture medium did not stimulate corneal epithelial closure in organ cultures. In their system, unlike ours, epithelial cells were scraped off the cornea, then the corneal blocks were excised and cultured; the epithelial cells migrated over this scraped surface, on which some basement membrane proteins probably remained. The rate of e ithelial migration in such a s stem mi ht be so fast t at the stimulatory effects of & or E EGg could not be detected. In our system, the corneal e ithelial cell layer migrated down the cut stromal sur ace of the corneal block where basement membranes could not be present. Therefore, the stimulatory effects of FN or EGF could be isolated. Their system more closely resembles the situation in vivo and is thus suitable for the investigation of normal, ra id epithelial closure. Our system may be more suitab e for more basic research regarding the causes of delayed epithelial wound healing, which has considerable clinical significance. The present results, obtained in an organ culture model, cannot be direct1 applied to the clinical situation. However, we have l Kemonstrated that even though both FN and EGF stimulate the healing of corneal epithelial defects, their actions are definitely different. FN appears to serve to maintain a suitable extracellular environment in which epithelial migration can occur, whereas EGF apparently acts as a trigger for the
R
E
R
I
I
0
1
3
10
30
100
concentration of serum or antiserum (P g/ml)
Fig. 7 . Effects of various concentrations of anti-EGF antiserum and EGF on epithelial migration. 0 en circle: anti-EGF antiserum, solid circle: anti-EGF antiserum andkGF (3 n iml) open triangle: normal rabbit serum, and solid triangle: normafrabbit serum and EGF (3 ngiml). Bars show SEM of 6 measurements.
have been investigated (Nishida et al., 198313, 1985; Deuel, 1987; Nishida, 1988). However, the precise mechanisms of their action, articularly of EGF, have not been completely clarifiec! The present results show a direct correlation between the length of the path of the corneal epithelium and the duration of exposure to FN. Although FN exhibited stimulatory action when added at any time during the 24-hour cultivation period, its continuous presence was required for maximum stimulatory effect. These observations are consistent with the binding of FN to integrin, leading to reorganization of the intracellular cytoskeleton (e.g., actin) in preparation for the active phase of epithelial mi ation or spreading (Buck and Horwitz, 1987). Indee , Nakagawa et al. (19851, using an immunof luorescence technique, reported the reorganization of cytoskeletal microfilaments in FN-stimulated migrating corneal epithelium. Corneal epithelial cells pre ared from the normal intact cornea did not respond a n 8 spread on a FN matrix demonstrating reduced activity to FN; however, when the cells were cultured for 12 hours or more, they spread on the FN matrix, demonstrating the ex ression of receptor activity (Nakagawa et al., 1990). lipurthermore, Nishida and Nakagawa (1989) showed that only corneal epithelial cells regenerating after mechanical debridement exhibit receptor activit for FN; from completely hea ed duced receptor activity. Thus, the receptors in the corneal epithelial closely associated with epithelial wound healing (Nishida and Naka awa, 1989). Unlike FN, EG was required to be present for certain period of time to exhibit its action. Once the EGF receptors in the corneal epithelial cells (Frati et al., 1972)bind EGF, the continuous resence of EGF is not required. As has been reporte in many other cells (Deuel, 1987), the EGF-EGF receptor complex is internalized, disappearing from the cell surface and leaving cells insensitive to EGF. Extracellular FN rovides a temporary matrix on which corneal epithe ial cells attach, spread, and mi-
cg.
Y
f
B
P
R
a
fi
R
P
P
NISHlDA ET AL
554
initiation of cell division and action of FN. In addition to FN and EGF, many growth factors, cytokines, and extracellular matrix roteins may be involved in corneal epithelial woun healing. Future investigation is required to clarify the orchestrated interactions of these agents.
cp
ACKNOWLEDGMENTS We would like to thank Dr. B. Horowitz of the New York Blood Center, New York, NY for the kind gift of human plasma FN and for the critical reading of our manuscript. We also thank Earth Pharmaceutical Co. for the supply of recombinant human EGF and polyclonal antiserum against human EGF. This research was supported in part by a ant from the Ministry of Education, Culture and cience of Japan and by a grant from Osaka Eye Bank, Osaka, Japan.
E
LITERATURE CITED Arturson, G. (1984)Epidermal growth factor in the healing of corneal wounds, epidermal wounds and partial-thickness scalds. Scand. J. Plast. Reconstr. Surg., 1833-37. Buck, C.A., and Horwitz, A.F. (1987) Cell surface receptors for extracellular matrix molecules. Annu. Rev. Cell Biol., 3:179-205. Cohen, S. (1962) Isolation of a mouse submaxillary gland protein acceleratin incisor eruption and eyelid opening in the new-born animal. J. kol. Chem., 237:1555-1562. DaGele, S., Frati, L., Fiore, C., and Santoni, G. (1979)The effect,ofthe epidermal growth factor (EGF) on the corneal epithelium in humans. Albrecht v. Graefes Arch. Klin. Exp. Ophtahl., 2103159-165. Deuel, T.E. (1987) Polypeptide growth factors: Roles in normal and abnormal cell growth. Annu. Rev. Cell Biol., 3:443492. Frati, L., Daniele, S., Delago, A., and Covelli, I. (1972) Selective binding of the epidermal growth factor and its specificeffects on the epithelial cells of the cornea. Exp. Eye Res., 14:135-141. Ho, P.C., Davis, W.H., Elliott, J.H., and Cohen, S. (1974) Kinetics of corneal epithelial regeneration and epidermal growth factor. Invest. Ophthalmol. 13:804-809. Hynes, R. (1985) Molecular biology of fibronectin. Annu. Rev. Cell Biol., 1:67-90. Kenyon, K.R. (1987) Morphology and pathological responses of the cornea to disease. In: The Cornea: Scientific Foundations and Clinical Practice, 2nd Edition. G. Smolin and R.A.Thoft, eds. Little, Brown and Company, Boston, pp. 63-98. Nakagawa, S., Nishida, T., and Manabe, R. (1985)Actin organization in migrating corneal epithelium of rabbits in situ. Exp. Eye Res., 41:335-343. Nakagawa, S., Nishida, T., Kodama, Y., and Itoi, M. (1990)Spreading of cultured corneal epithelial cells on fibronectin and other extracellular matrices. Cornea, 9:125-130. Nishida, T. (1988) Role of fibronectin in the corneal epithelial wound healing. In: The Cornea: Transactlons of the World Congress on the Cornea 111. H. D. Cavanagb, ed. Raven Press, New York, pp. 619-625. Nishida, T., and Nakagawa, S. (1989) Expression of fibronectin receptors in corneal epithelial cells. In: Healing Processes in the Cornea. R.W. Beuerman, C.E. Crosson, and H.E. Kaufman, eds. PortfolioPublishing Company, The Woodlands,Vol. 1, p 127 135 Nishida, T., Nakagawa, S., Awata, T., Ohashi, Y.,Watanate, K, and
Manabe, R. (1983a) Fibronectin promotes epithelial migration of cultured rabbit cornea in situ. J. Cell Biol., 971653-1657. Nishida, T., Ohashi, Y., Awata, T., and Manabe, R. (1983b)Fibronectin: A new therapy for corneal trophic ulcer. Arch. Ophthalmol., 101:104&1048. Nishida, T., Nakagawa, S., Nishibayashi, C., Tanaka, H., and Manabe, R. (1984a) Fibronectin enhancement of corneal epithelial wound healinn of rabbits in vivo. Arch. ODhthalmol.,102:455.456. Nishida, T., Ta;aka, H., Nakagawa, S., Sisabe, T., Awata, T., and Manabe, R. (198413) Fibronectin synthesis by the rabbit cornea: Effects of mouse epidermal growth factor and cyclic AMP analogs. J n J Ophthalmol., 28196-202. Nis\ida;T., Nakagawa, S., and Manabe, R. (1985)Clinical evaluation of fibronectin eyedrops on epithelial disorders after herpetic keratitis. Ophthalmology, 92:213-216. Nishida, T., Naka awa, S., Watanabe, K., Yamada, K.M., Otori, T., and Berman, (1988) A peptide from fibronectin cell-bindin domain inhibits attachment of epithelial cells. Invest. Ophthalmof Vis. Sci., 291820-1825. Ohashi, Y., Motokura, M., Kinoshita, Y., Mano, T., Watanabe, H., Kinoshita, S., Manabe, R., Oshiden, and Yanaihara, C. (1989) Presence of epidermal growth factor in human tears. Invest. Ophthalmol. Vis. Sci., 30:1879-1882. Petroutsos, G., Courty, J., Guimaraes, R., Pouliquen, Y., Barritault, D., Plouet, J., and Courtois, Y. (1984) Comparison of the effects of EGF, pFGF, and EDGF on corneal epithelium would healing. Curr. Eye Res., 3:593-598. Reim, M., Busse, S., Leber, M., and Schulz, C. (1988) Effect of e idermal growth factor in severe experimental alkali burns. OphtEalmic Res.. 20:327-331. Ruoslahti, E. (1981)Fibronectin; Current concepts of its structure and function. Coll. Res., 1:95-128. Savage, C.R., Jr., and Cohen, S. (1973) Proliferation of corneal epithelium induced by epidermal growth factor. Exp. Eye Res., -
1
MS.
v.,
-1.5c%l-366. - .- - - - ...
Schultz, G., Davis, J., and Eiferman, R. (1988a) Growth factors and corneal e ithelium. Cornea, 7:9C101. Schultz, G.{., Woost, P.G., and Eiferman, R.A. (1988b)Modificationof corneal wound healing by growth factors. In: The Cornea: Transactions of the World Congress on the Cornea 111.H.D. Cavanagh, ed. Raven Press, New York, pp. 15-21. Singh, G., and Foster, C.S. (1987) E idermal growth factor in alkaliburned corneal epithelial wouncf healing. Am. J. Ophthalmol., 103:802-807. Soong, H.K., Hassan, T., Varani, J., Huang, S.C.M., and Brennan, M. (1989a) Fibronectin Dose not enhance epidermal growth factormediated acceleration of corneal epithelial wound closure. Arch. Ophthalmol., 1071052-1054. Soong, H.K., McClenic, B., Varani, J.,Hassan, T., Huang, S.C.M.,and Brenz, R. (1989b) EGF does not enhance corneal epithelial cell motility. Invest. Ophthalmol. Vis. Sci., 30:1808-1812. Spigelman, A.V., Vernot, J.A., and Deutsch, T.A. (1985) Fibronectin in alkali bums of the rabbit cornea. Cornea, 4:16%172. Tripathi, B.J., Kwait, P.S., and Tripathi, R.C. (1990) Corneal growth factors: A new generation of ophthalmic pharmaceuticals. Cornea, 9:2-9. Watanabe, K., Nakagawa, S., and Nishida, T. (1987) Stimulatory effects of fibronectin and EGF on migration of corneal epithelial cells. Invest. Ophthalmol. Vis. Sci., 28205211. Watanabe, K., Nakagawa, S., and Nishida, T. (1988)Chemotacticand haptotactic activities of fibronectin for cultured rabbit corneal epithelial cells. Invest. Ophthalmol. Vis. Sci., 29:572-577. Yamada, K.M. (1983) Cell surface interactions with extracellular materials. Annu. Rev. Biochem., 52:761-799.
Differential Modes of Action of Fibronectin and Epidermal Growth Factor on Rabbit Corneal Epithelial Migration TERUO NISHIDA,' MASATSUGU NAKAMURA, HlROSHl MISHIMA, AND TOSHlFUMl OTORI Department of Ophtha!mology, Kinki University School of Medicine, Osaka-Sayama City, Osaka 589, japan In order to clarify the roles of fibronectin (FN) and epidermal growth factor (EGF) in corneal wound healing, we cultured blocks of excised rabbit cornea for 24 hours in media containing one of these agents, then measured the length of the path of the epithelial layer that had migrated down the side of the block. Both FN and EGF stimulated epithelial migration significantly in a dose-dependentfashion. Responses to EGF involved a time lag of at least 12 hours before stimulation could be observed, but there was no lag-time for FN-stimulated migration. FN was maximally effective only if it was continuously present. In contrast, exposure to ECF for 6 hours did not stimulate epithelial migration, but exposure for 9 hours resulted in the same stimulatory effects as were observed after 24 hours' continuous exposure. Anti-FN antibody inhibited the FN- and EGF-stimulated migration of corneal epithelium. But anti-EGF antibody inhibited only EGFstimulated migration and had no effect on FN-stimulated migration. These results indicate that, unlike FN, EGF need not be present, once the epithelial cells have recognized its signal. Furthermore, the stimulatory effect of EGF depended on FN, while that of FN was independent of EGF. The effects of EGF on migration of corneal epithelium may, therefore, be mediated by FN.
The cornea consists of epithelium, stroma, and endothelium. The stroma has a regularly layered structure of collagen fibers and proteoglycans, such as keratan sulfate (Kenyon, 1987). An amorphous condensation of collagen forms the Bowman's membrane, on which the basement membrane of the epithelium rests. Basal cells rest on this basement membrane, whose extracellular matrix proteins, fibronectin (FN), laminin, and type IV collagen help to maintain the layered, striated structure of the corneal e ithelium. Maintenance of an intact corneal epithelia layer is an important defense a ainst infection, but the direct contact of the cornea wit the external environment often results in disturbance of the smooth corneal epithelium. The immediate resurfacing of orchestrated responses extracellular factors, cellular matrix roteins. Among the re orted factors that modulate t is migration of epitherium to cover defects, FN and epidermal growth factor (EGF) have been studied the most (Nishida, 1988; Tripathi et al., 1990).EGF binds to corneal epithelial cells (Fratiet al., 1972; Ho et al., 1974), stimulating wound closure (Savage and Cohen, 1973; Arturson, 1984; Petroutsos et al., 1984; h i m et al., 1988; Schultz et al., 1988a,b). The presence of EGF in tears was recently reported (Ohashi et al., 1989). FN and EGF both stimulate the migration of e ithelium in cultured rabbit cornea and facilitate the c osure of corneal epithelial defects in vivo (Frati et al., 1972; Savage and Cohen, 1973; Daniele
P
a
R
P
0 1990 WILEY-LISS, INC.
et al., 1979; Nishida et al., 1983a, 1984a; Arturson, 1984; Petroutsos et al., 1984; S igelman et al., 198.51, but their modes of action di fer (Ho et al., 1974; Watanabe et al., 1987). FN is chemotactic and haptotactic for corneal epithelial cells, but EGF is not (Watanabe et al., 1988). We previously reported that FN stimulated the attachment and spreading of cultured, single rabbit corneal epithelial cells on a FN matrix. Furthermore, FN stimulated migration of rabbit corneal e ithelium as a sheet in or an culture (Nishida et ay., 1983a) and in vivo (dshida et al., 1984a). The mechanism of the stimulatory effects of FN might be based on the adhesive characteristics of the FN molecule. Each domain of the FN molecule exhibits specific binding activity for collagen, fibrin, heparin, or its cellular receptor, known as inte in (Ruoslahti, 1981; Yamada, 1983; Hynes, 1985; Buc and Horwitz, 1987). FN is thought to bridge the cellular FN receptor and underlying extracellular matrix materials such as collagen. Indeed, the addition of GRGDS, the amino acid sequence of the cell-binding domain of FN, inhibited attachment and spreading demonstrating the presence of a FN receptor (integrin) in corneal epithelial cells (Nishida et al., 1988). Since the first report of Cohen (Cohen, 1962), the stimulatory effects of EGF have been studied extensively. Frati
F
iT
Received May 17, 1990; accepted September 6, 1990. 'To whom reprint requestskorrespondence should be addressed.
550
NISHIDA ET AL.
et al. (Frati et al., 1972) re orted that EGF rece tors are present in corneal epit elial cells and that GF stimulated DNA and RNA synthesis in corneal epithelial cells. Topical application of EGF was shown to stimulate reepithelization of various types of corneal epithelial defects (Daniele et al., 1979; Petroutsos et al., 1984; Singh and Foster, 1987; Schultz et al., 1988a,b). EGF stimulated the migration of corneal epithelium down the cut stromal surface of corneal blocks, apparently by stimulating cell proliferation, as measured by the incorporation of 3H-th midine (Watanabe et al., 1987). On the other hand, N does not stimulate DNA synthesis (Watanabe et al., 1987; Nishida et al., 1988). Diverse pathological conditions can lead to persistent epithelial defects, with or without ulceration of the corneal stroma. Use of FN and EGF for the treatment of such disorders is being investi ated. On a thin section taken from a block of culture cornea, epithelial migration can be visualized as a heavily stained cell layer extending art way down the side of the block, from what was t e anterior surface of the eye toward the endothelium. We measured the length of the path of the epithelium, which we refer to as the migration of the epithelium, on the blocks cultured with FN or EGF to determine whether the continuous presence of these agents is required for their maximum effect. We studied also the effects of antibodies against FN and EGF on this migration of epithelium. Information gained from our studies may be useful in optimizing the clinical application of these agents and in differentiating their use for different clinical sta es of persistent corneal epithelial defects, whose mec anisms of action are not completely understood. MATERIALS AND METHODS Albino rabbits wei hing 2 to 3 kg were obtained from Awazu Experimenta Animals (Settsu, Osaka, J a an), phosphate-buffered saline (PBS) and TC-199 cu ture medium were from the Research Foundation for Microbial Diseases of Osaka University (Suita, Osaka, Japan), and plastic multi-well culture dishes (24 wells, #3424) were from Costar (Cambrid e, MA). Human plasma FN was from the New York B ood Center (New York, NY). Recombinant human EGF and polyclonal anti-human EGF rabbit antiserum were from Earth Pharmaceutical (Akoh, H ogo, Japan). The purity of FN and EGF were assesse by SDS polyacrylamide gel electrophoresis and by high-performance liquid chromatography. Guinea pig antiserum against rabbit plasma FN was prepared as characterized and described previously (Nishida et al., 1983a). Polyacrylamide gel electrophoresis and Western blottin indicated that the antibodies used were specific or FN or EGF. Rabbit corneas were cultured as described previously, with sli ht modification (Nishida et al., 1983a; Watanabe et a f , 1987). Rabbits were sacrificed with an intravenous overdose of pentobarbital. Eyes were enucleated; a sclerocorneal section was excised, washed several times with sterile PBS, and cut into 2 x 4-mm blocks with a razor blade. The blocks were cultured in plastic multi-well tissue culture dishes with 1 ml of serum-free TC-199 culture medium with the experimental agents (human plasma FN, recombinant hu-
R
E
P
%
R
a
P
P
f
B
f?
man EGF, or antisera). Cultures were maintained at 37°C in a humidified incubator under 5%COz.The rate of epithelial mi ration was determined by examinin one series of cu tures after 6, 12, 18, or 24 hours. A1 oiher cultures were maintained for 24 hours; in the first of these experiments, the culture medium containing FN (100 kg/ml) was re laced by serum-free TC-199 medium after 6,12, or 18 ours; other specimens were cultured without FN for the first 12 hours and with FN for the last 12 hours. Another group of corneal blocks was incubated with FN for the entire 24 hours. The culture medium containing EGF (3 nglml) was replaced by serum-free TC-199 after 3, 6, 9, or 12 hours, and other groups of corneal blocks were cultured without EGF for the first 12 hours and with EGF for the last 12, or with EGF for the entire 24 hours. Control specimens were cultured in serum-free TC-199 medium alone. In the second series of experiments, s ecimens were cultured with various concentrations of N (1,10, and 100 p,g/ml) or EGF (0.3, 1,3, and 10 nglml) for 6 or 12 hours, and then the culture medium was replaced by serum-free TC-199 medium. Others were cultured at the same concentrations for the entire 24 hours. The third series of experiments employed the following agents: FN (100 kg/ml) with normal guinea pig serum, anti-FN antiserum (0.5,1.0,2.0,and 5.0 mg/ml) anti-FN anti-serum (at the same concentrations) with FN or with EGF (3 nglml) or normal uinea pig serum (control). Other cultures contained E F (3 ng/ml) with normal rabbit serum, anti-EGF anti-serum (1,3,10,30, or 100 Kgiml), anti-EGF antiserum (at the same concentrations) with EGF or FN (100 pg/rnl), or normal rabbit serum (control). Epithelial migration down the cut side of the block, toward the endothelial side, was measured as reported previously (Nishida et al., 1983a; Watanabe et al., 1987). In brief, the cultivated corneal blocks were washed with PBS and fixed overnight with a mixture of 100% ethanol and glacial acetic acid (955) at 4°C. The blocks were deh drated, immersed in xylene, and embedded in para in; three 4-km sections were cut, 300 bm apart, from each block, so that elon ation of the epithelial path could be determined. After deparaffinization, the specimens were stained with hematoxylin-eosin and observed under a light microsco e (Model BH-2, 01 mpus, Tok 0 , Japan). Photogra were taken with lack and w ite film (Neopan F, 8uji Films, Tokyo, J a an) with the aid of an automatic exposure meter. e length of the epithelial cell layer was measured on the photographs of each corneal block. Results were expressed as means ( * SEMI of 6 measurements. Statistical analysis was performed by Student's t-test. RESULTS Rates of migration of the epithelium are shown in Figure 1. The corneal epithelium cultured in serumfree TC-199 medium migrated as a sheet over the cut surface of the corneal stroma; the len h of the migrated epithelium did not increase signi icantly during the first 6 hours, but active migration was noted between hours 12 and 24 (P< .001). Epithelium cultured with FN (100 Fg/ml) also migrated slightly during the first 6 hours, and more rapidly thereafter,
I
K
K
i
8
H
B &
1s
l
8"
551
FN AND EGF ON CORNEAL EPITHELIAL MIGRATION
- 750
-
TABLE 1. Duration of exposure to F N or EGF related to corneal epithelial migration'
E
1
v
.-c 9 .-0
Exposure
500 -
period
FN (100pg/ml) W
(hr)
(rm)
None
-E
394+ 15 520+23* 4 3 0 f 27 431 *29
0-24 0-12 12-24
2ml 250 -
5 .n m
EGF (3ng/ml) Migration %I (wm) 389 f 23 100 579 f 44* 149 147 570 f 40*
Migration
100 132 109 109
377 f 30
97
* SEM (n = 6). * P < ,005 against controls. 'Means
00
6
12
18
24
hours in culture (hr)
Fig. 1. Chronological changes in the length of the path of corneal epithelium cultured in unsupplemented TC-199 (open circle), in TC-199 containing FN (100 pgiml, solid circle), and in TC-199 containing EGF (3 ngiml, open triangle). Bars show SEM of 6 measurements.
fF
but FN si ificantly increased the len h of the epithelial cel layer at 24 hours, compare with controls (P< ,001).In the presence of EGF (3 ng/ml), the length of 12 hours was increased only slightly compared with controls. After 24 hours, however, the length of the path was about 50% greater than that of specimens cultured in unsupplemented TC-199 medium (P < .001). These results also demonstrated two phases in the epithelial migration: a preparatory lag phase and an active migratory phase. The effect of the presence of FN (100 pg/ml) or EGF (3 nglml) during the first or the last 12 hours of the 24-hour incubation period is summarized in Table 1. When the corneal blocks were cultured in unsupplemented TC-199 culture medium, the length was 394 or 389 km. The continuous presence of FN or EGF increased the length of the path of 520 (P < .005) or 579 (P < .005) pm, respectively. Exposure to FN for the first or for the last 12 hours resulted in increases which were similar, and intermediate in magnitude between those seen when medium was unsupplemented or when FN was present for 24 hours. In contrast, exposure to EGF for the first 12 hours resulted in as much epithelial migration as that seen in specimens exposed to EGF continuously for 24 hours, whereas exposure to EGF for the last 12 hours did not stimulate the migration of epithelium at all. Thus, FN stimulated epithelial migration whether added early or late, whereas EGF produced an observable response only after it had been resent for a certain period of time. Because the thic ness of the cultured cornea was limited, we could not continue the cultivation beyond 24 hours. Thus it is difficult to conclude whether or not EGF would be effective if present during the late phase, when active migration was observed. The length of the migrated epithelium after 24 hours was proportional to the duration of exposure to FN (6, 12,18,or 24 hours) (Fig. 2). Response to EGF was more com licated; exposure to EGF for up to 6 hours did not resu t in any increase in epithelial migration, but exposure for 9 or 12 hours resulted in path lengths similar to those seen on corneal blocks cultured continuously with EGF for 24 hours (Fig. 2).
Y
R
P
* 14
0
500
250
** 150
epithelial migration (pm)
Fig. 2. Effects of the duration of exposure to FN (100 p iml, open column) or EGF (3 ng/ml, solid column) on the length of t i e path of corneal epithelium in corneal blocks cultured for 24 hours. Bars show SEM of 6 measurements. *P< .02 and **P< ,005against control.
When we tested whether these effects depended on the concentrations of FN and EGF, we found, in the case of FN, that the length of the path increased with the concentrations (1, 10, or 100 pg/ml), whether exposure had been for 6, 12, or 24 hours. As expected, the increases in the length of the epithelium after incubation with FN for 6 or 12 hours were less than those seen after incubation for 24 hours, even at the highest concentrations (Fig. 3). The length of the path of the corneal epithelium showed almost identical, concentration-dependent increases when blocks were cultured with various concentrations of EGF for 12 or 24 hours. However, no increase in len h was observed when the corneal blocks were culturec? for 6 hours with EGF, even at its highest concentration (10 ng/ml) (Fi . 4). hgure 5 shows the quantitative effects of FN and various concentrations of anti-FN antiserum. The addition of FN alone increased the corneal epithelial migration significant1 (P < .05). Anti-FN antiserum alone inhibited epithe ial migration significantly, in a dose-dependent manner, apparently abolishing the effects of endogenous FN at the higher concentrations (P< .001 at 5 mg/ml of anti-FN antiserum). The addition of anti-FN antiserum a t a concentration of 0.5 mg/ml completely reversed the stimulatory effect of
P
552
NISHIDA ET AL
750
1
E
a
I
1
0
10
100
0
concentration of FN (u g/ml)
r
1
I
I
3
10
concentration of EGF (ng/ml)
Fig. 3. Effects of the concentration of FN on the length of the path of corneal epithelium in corneal blocks exposed for 6 (0 en circle), 12 (solid circle), or 24 (open triangle) hours to FN. Bars siow SEM of 6 measurements.
750
0. 3
Fig. 4. Effects of the concentration of EGF on the length of the path of corneal epithelium in corneal blocks exposed for 6 (open circle), 12 (solid circle), or 24 (open triangle) hours to EGF. Bars show SEM of 6 measurements.
r
750 +,----~~---........_..__._.__.__~ ___.....___........ ..--.__..... -4
T
I
I
0
0. 5
1
5
2
0
0. 5
1
2
5
concentration of serum or antiserum (mg/ml)
concentration of serum or antiserum (mglml) Fig. 5. Effects of various concentrations of anti-FN antiserum and FN on the length of the path of epithelial migration after 24 hours. Open circle: anti-FN antiserum, solid circle: anti-FN antiserum and FN (100 pg/ml), open triangle: normal guinea pig serum, and solid triangle: normal guinea pig serum and FN (100 pg/ml). Bars show SEM of 6 measurements.
Fig. 6. Effects of various concentrations of anti-FN antiserum and EGF on epithelial migration. Open circle: anti-FN antiserum, solid circle: anti-FN antiserum and EGF (3 ngiml), open triangle: normal guinea pig serum, and solid triangle: normal guinea pig serum and EGF (3 ng/ml). Bars show SEM of 6 measurements.
exogenous FN (at 0.5 m /ml, P < .05, different from the group cultured with F alone; at 5.0 mg/ml, P < .001, different from the group cultured with FN alone). No statistically significant difference was observed between these specimens and those cultured with anti-FN antiserum alone. Non-immune guinea ig normal serum did not appear to influence epithefial migration. These results demonstrated that FN mediated migration of the corneal epithelium. Figure 6 shows the effects of various concentrations of anti-FN antiserum in the presence or absence of EGF. The presence of non-immune guinea pig normal serum did not affect e ithelial migration in the presence or absence of E F. The addition of EGF alone increased the length of the path of the corneal e ithelium significantly (P < .05), but the addition o antiFN antiserum (0.5 mg/ml) reversed this EGF-stimulated increase completely (P< .02). The addition of anti-FN antiserum inhibited epithelial migration in a dose-dependent fashion (at 5 mg/ml of anti-FN antiserum, P < .01 compared with specimens cultured with-
out EGF; P < ,001 compared with specimens cultured EGF). with __ Next we-investi ated the effects of various concentrations of anti-E F antiserum. The addition of antiEGF antiserum or rabbit normal serum affected neither epithelial migration nor the stimulatory effect of FN at any antiserum concentrations examined (data not shown). However, anti-EGF antiserum reversed the EGF-stimulated mi ration of the epithelium in a dosedependent fashion ( ig. 7).
If
8
P
~~~~
8
%
DISCUSSION Because a smooth, intact corneal epithelium is essential for the maintenance of good vision and defense of the cornea against infection, it is ver important to understand the mechanism(s1 that regu ate the resurfacing of epithelial defects. Recent advances in cell biology allow us to investigate the role of extracellular matrix and growth factors in corneal wound healing. The clinical efficacies of both FN and EGF for the treatment of non-healing corneal epithelial defects
T
553
FN AND EGF ON CORNEAL EPITHELIAL MIGRATION
750 r
grate. As we previously re orted, the addition of mouse EGF stimulated the synt esis and secretion of FN in cultured full-thickness rabbit cornea (Nishida et al., 1984b),indicating that EGF stimulates the synthesis of FN. FN secreted into the extracellular s ace may bind to cell surface receptors for FN, and t us stimulate epithelial migration, although it has not been clarified yet which kinds of corneal cells are responsible for FN synthesis. Recently, we found that the number of cells attached to the FN matrix increased when the corneal e ithelial cells were cultured with EGF, suggesting t at EGF might stimulate the expression of FN receptors by the corneal epithelial cells (unpublished data). Therefore EGF might act on the cornea in a combination of different ways: by increasing e ithelial cell numbers (proliferation), by increasin N synthesis # stimulating and secretion by the whole cornea, anc fby expression of FN receptors. The addition of anti-FN antiserum inhibited stimulation of epithelial migration by either FN or EGF. Furthermore anti-FN inhibited epithelial migration that would take lace without the addition of exogenous FN. The adfition of anti-EGF antiserum did not affect epithelial migration driven by FN, but did inhibit the stimulatory action of exo enous EGF. In the preliminary experiments, when t e higher concentrations of anti-FN was added, most of the epithelial cells detached from each other. Although anti-FN used in this study was not affinity purified, we noted that normal guinea pig serum did not affect epithelial migration and that anti-EGF. These findings suggest that the inhibition of normal e ithelial migration, in the absence of exo enous FN, is LTue solely to antiserum directed against N. These results demonstrate that the action of EGF on corneal epithelial cells might be mediated through FN. Recently Soong and his associates (1988a,b) reported that the addition of EGF or FN to the culture medium did not stimulate corneal epithelial closure in organ cultures. In their system, unlike ours, epithelial cells were scraped off the cornea, then the corneal blocks were excised and cultured; the epithelial cells migrated over this scraped surface, on which some basement membrane proteins probably remained. The rate of e ithelial migration in such a s stem mi ht be so fast t at the stimulatory effects of & or E EGg could not be detected. In our system, the corneal e ithelial cell layer migrated down the cut stromal sur ace of the corneal block where basement membranes could not be present. Therefore, the stimulatory effects of FN or EGF could be isolated. Their system more closely resembles the situation in vivo and is thus suitable for the investigation of normal, ra id epithelial closure. Our system may be more suitab e for more basic research regarding the causes of delayed epithelial wound healing, which has considerable clinical significance. The present results, obtained in an organ culture model, cannot be direct1 applied to the clinical situation. However, we have l Kemonstrated that even though both FN and EGF stimulate the healing of corneal epithelial defects, their actions are definitely different. FN appears to serve to maintain a suitable extracellular environment in which epithelial migration can occur, whereas EGF apparently acts as a trigger for the
R
E
R
I
I
0
1
3
10
30
100
concentration of serum or antiserum (P g/ml)
Fig. 7 . Effects of various concentrations of anti-EGF antiserum and EGF on epithelial migration. 0 en circle: anti-EGF antiserum, solid circle: anti-EGF antiserum andkGF (3 n iml) open triangle: normal rabbit serum, and solid triangle: normafrabbit serum and EGF (3 ngiml). Bars show SEM of 6 measurements.
have been investigated (Nishida et al., 198313, 1985; Deuel, 1987; Nishida, 1988). However, the precise mechanisms of their action, articularly of EGF, have not been completely clarifiec! The present results show a direct correlation between the length of the path of the corneal epithelium and the duration of exposure to FN. Although FN exhibited stimulatory action when added at any time during the 24-hour cultivation period, its continuous presence was required for maximum stimulatory effect. These observations are consistent with the binding of FN to integrin, leading to reorganization of the intracellular cytoskeleton (e.g., actin) in preparation for the active phase of epithelial mi ation or spreading (Buck and Horwitz, 1987). Indee , Nakagawa et al. (19851, using an immunof luorescence technique, reported the reorganization of cytoskeletal microfilaments in FN-stimulated migrating corneal epithelium. Corneal epithelial cells pre ared from the normal intact cornea did not respond a n 8 spread on a FN matrix demonstrating reduced activity to FN; however, when the cells were cultured for 12 hours or more, they spread on the FN matrix, demonstrating the ex ression of receptor activity (Nakagawa et al., 1990). lipurthermore, Nishida and Nakagawa (1989) showed that only corneal epithelial cells regenerating after mechanical debridement exhibit receptor activit for FN; from completely hea ed duced receptor activity. Thus, the receptors in the corneal epithelial closely associated with epithelial wound healing (Nishida and Naka awa, 1989). Unlike FN, EG was required to be present for certain period of time to exhibit its action. Once the EGF receptors in the corneal epithelial cells (Frati et al., 1972)bind EGF, the continuous resence of EGF is not required. As has been reporte in many other cells (Deuel, 1987), the EGF-EGF receptor complex is internalized, disappearing from the cell surface and leaving cells insensitive to EGF. Extracellular FN rovides a temporary matrix on which corneal epithe ial cells attach, spread, and mi-
cg.
Y
f
B
P
R
a
fi
R
P
P
NISHlDA ET AL
554
initiation of cell division and action of FN. In addition to FN and EGF, many growth factors, cytokines, and extracellular matrix roteins may be involved in corneal epithelial woun healing. Future investigation is required to clarify the orchestrated interactions of these agents.
cp
ACKNOWLEDGMENTS We would like to thank Dr. B. Horowitz of the New York Blood Center, New York, NY for the kind gift of human plasma FN and for the critical reading of our manuscript. We also thank Earth Pharmaceutical Co. for the supply of recombinant human EGF and polyclonal antiserum against human EGF. This research was supported in part by a ant from the Ministry of Education, Culture and cience of Japan and by a grant from Osaka Eye Bank, Osaka, Japan.
E
LITERATURE CITED Arturson, G. (1984)Epidermal growth factor in the healing of corneal wounds, epidermal wounds and partial-thickness scalds. Scand. J. Plast. Reconstr. Surg., 1833-37. Buck, C.A., and Horwitz, A.F. (1987) Cell surface receptors for extracellular matrix molecules. Annu. Rev. Cell Biol., 3:179-205. Cohen, S. (1962) Isolation of a mouse submaxillary gland protein acceleratin incisor eruption and eyelid opening in the new-born animal. J. kol. Chem., 237:1555-1562. DaGele, S., Frati, L., Fiore, C., and Santoni, G. (1979)The effect,ofthe epidermal growth factor (EGF) on the corneal epithelium in humans. Albrecht v. Graefes Arch. Klin. Exp. Ophtahl., 2103159-165. Deuel, T.E. (1987) Polypeptide growth factors: Roles in normal and abnormal cell growth. Annu. Rev. Cell Biol., 3:443492. Frati, L., Daniele, S., Delago, A., and Covelli, I. (1972) Selective binding of the epidermal growth factor and its specificeffects on the epithelial cells of the cornea. Exp. Eye Res., 14:135-141. Ho, P.C., Davis, W.H., Elliott, J.H., and Cohen, S. (1974) Kinetics of corneal epithelial regeneration and epidermal growth factor. Invest. Ophthalmol. 13:804-809. Hynes, R. (1985) Molecular biology of fibronectin. Annu. Rev. Cell Biol., 1:67-90. Kenyon, K.R. (1987) Morphology and pathological responses of the cornea to disease. In: The Cornea: Scientific Foundations and Clinical Practice, 2nd Edition. G. Smolin and R.A.Thoft, eds. Little, Brown and Company, Boston, pp. 63-98. Nakagawa, S., Nishida, T., and Manabe, R. (1985)Actin organization in migrating corneal epithelium of rabbits in situ. Exp. Eye Res., 41:335-343. Nakagawa, S., Nishida, T., Kodama, Y., and Itoi, M. (1990)Spreading of cultured corneal epithelial cells on fibronectin and other extracellular matrices. Cornea, 9:125-130. Nishida, T. (1988) Role of fibronectin in the corneal epithelial wound healing. In: The Cornea: Transactlons of the World Congress on the Cornea 111. H. D. Cavanagb, ed. Raven Press, New York, pp. 619-625. Nishida, T., and Nakagawa, S. (1989) Expression of fibronectin receptors in corneal epithelial cells. In: Healing Processes in the Cornea. R.W. Beuerman, C.E. Crosson, and H.E. Kaufman, eds. PortfolioPublishing Company, The Woodlands,Vol. 1, p 127 135 Nishida, T., Nakagawa, S., Awata, T., Ohashi, Y.,Watanate, K, and
Manabe, R. (1983a) Fibronectin promotes epithelial migration of cultured rabbit cornea in situ. J. Cell Biol., 971653-1657. Nishida, T., Ohashi, Y., Awata, T., and Manabe, R. (1983b)Fibronectin: A new therapy for corneal trophic ulcer. Arch. Ophthalmol., 101:104&1048. Nishida, T., Nakagawa, S., Nishibayashi, C., Tanaka, H., and Manabe, R. (1984a) Fibronectin enhancement of corneal epithelial wound healinn of rabbits in vivo. Arch. ODhthalmol.,102:455.456. Nishida, T., Ta;aka, H., Nakagawa, S., Sisabe, T., Awata, T., and Manabe, R. (198413) Fibronectin synthesis by the rabbit cornea: Effects of mouse epidermal growth factor and cyclic AMP analogs. J n J Ophthalmol., 28196-202. Nis\ida;T., Nakagawa, S., and Manabe, R. (1985)Clinical evaluation of fibronectin eyedrops on epithelial disorders after herpetic keratitis. Ophthalmology, 92:213-216. Nishida, T., Naka awa, S., Watanabe, K., Yamada, K.M., Otori, T., and Berman, (1988) A peptide from fibronectin cell-bindin domain inhibits attachment of epithelial cells. Invest. Ophthalmof Vis. Sci., 291820-1825. Ohashi, Y., Motokura, M., Kinoshita, Y., Mano, T., Watanabe, H., Kinoshita, S., Manabe, R., Oshiden, and Yanaihara, C. (1989) Presence of epidermal growth factor in human tears. Invest. Ophthalmol. Vis. Sci., 30:1879-1882. Petroutsos, G., Courty, J., Guimaraes, R., Pouliquen, Y., Barritault, D., Plouet, J., and Courtois, Y. (1984) Comparison of the effects of EGF, pFGF, and EDGF on corneal epithelium would healing. Curr. Eye Res., 3:593-598. Reim, M., Busse, S., Leber, M., and Schulz, C. (1988) Effect of e idermal growth factor in severe experimental alkali burns. OphtEalmic Res.. 20:327-331. Ruoslahti, E. (1981)Fibronectin; Current concepts of its structure and function. Coll. Res., 1:95-128. Savage, C.R., Jr., and Cohen, S. (1973) Proliferation of corneal epithelium induced by epidermal growth factor. Exp. Eye Res., -
1
MS.
v.,
-1.5c%l-366. - .- - - - ...
Schultz, G., Davis, J., and Eiferman, R. (1988a) Growth factors and corneal e ithelium. Cornea, 7:9C101. Schultz, G.{., Woost, P.G., and Eiferman, R.A. (1988b)Modificationof corneal wound healing by growth factors. In: The Cornea: Transactions of the World Congress on the Cornea 111.H.D. Cavanagh, ed. Raven Press, New York, pp. 15-21. Singh, G., and Foster, C.S. (1987) E idermal growth factor in alkaliburned corneal epithelial wouncf healing. Am. J. Ophthalmol., 103:802-807. Soong, H.K., Hassan, T., Varani, J., Huang, S.C.M., and Brennan, M. (1989a) Fibronectin Dose not enhance epidermal growth factormediated acceleration of corneal epithelial wound closure. Arch. Ophthalmol., 1071052-1054. Soong, H.K., McClenic, B., Varani, J.,Hassan, T., Huang, S.C.M.,and Brenz, R. (1989b) EGF does not enhance corneal epithelial cell motility. Invest. Ophthalmol. Vis. Sci., 30:1808-1812. Spigelman, A.V., Vernot, J.A., and Deutsch, T.A. (1985) Fibronectin in alkali bums of the rabbit cornea. Cornea, 4:16%172. Tripathi, B.J., Kwait, P.S., and Tripathi, R.C. (1990) Corneal growth factors: A new generation of ophthalmic pharmaceuticals. Cornea, 9:2-9. Watanabe, K., Nakagawa, S., and Nishida, T. (1987) Stimulatory effects of fibronectin and EGF on migration of corneal epithelial cells. Invest. Ophthalmol. Vis. Sci., 28205211. Watanabe, K., Nakagawa, S., and Nishida, T. (1988)Chemotacticand haptotactic activities of fibronectin for cultured rabbit corneal epithelial cells. Invest. Ophthalmol. Vis. Sci., 29:572-577. Yamada, K.M. (1983) Cell surface interactions with extracellular materials. Annu. Rev. Biochem., 52:761-799.
