responsible for accelerated wound healing in the presence of EGF. Invest Ophthalmol Vis Sci. 30:1808-1812,1989. Almost immediately after corneal epithelial.
Investigative Ophthalmology & Visual Science, Vol. 30, No. 8, August 1989 Copyright © Association for Research in Vision and Ophthalmology
EGF Does Not Enhance Corneal Epithelial Cell Motility H. Kaz Soong,* Brian McClenict James Varani,t Tarek Hassan,* Samuel C. M. Huang,* and Richard Brenz" Although it is well known that epidermal growth factor (EGF) accelerates corneal epithelial wound healing by stimulating mitosis, it is also believed that EGF may directly stimulate the motility of individual corneal epithelial cells. We employed three different experimental methods to determine if EGF does indeed enhance the motility of corneal epithelial cells (independent of mitotic effects). First, the effects of EGF on the motility of tissue-cultured rat and rabbit corneal epithelial cells were investigated by a Boyden chamber assay. In rat corneal epithelium, these effects were further investigated by a second method, the agarose drop assay. Both assay techniques demonstrated no increase in corneal epithelial cell motility in the presence of EGF. These findings were corroborated by a third method which consisted of measuring the closure rate of epithelial wounds in organ-cultured rat corneas in the presence and absence of EGF, while concurrently arresting mitosis with colchicine. The wound closure rate before addition of any drug was 0.46 ± 0.03 mm2/hr. The wound closure rate with EGF (50 ng/ml) was 0.55 ± 0.03 mm2/hr, significantly {P < 0.005) more rapid than the drug-free controls. However, when EGF (50 ng/ml) and colchicine (40 fig/m\) were used simultaneously, the acceleration of wound closure by EGF was completely negated by the presence of colchicine, resulting in a wound closure rate (0.46 ± 0.06 mm2/hr) that did not differ significantly (P > 0.50) from that of the drug-free control. These results, together with the Boyden chamber and agarose drop assay results, suggest that the acceleration of corneal epithelial wound healing by EGF is due primarily to increased cell proliferation (with the increase in cell population causing the cells to spill over into the epithelial defect). It appears that individual cell motility, independent of mitosis, is not primarily responsible for accelerated wound healing in the presence of EGF. Invest Ophthalmol Vis Sci 30:1808-1812,1989
Almost immediately after corneal epithelial wounding, the surrounding cells migrate into the wound to cover the defect as rapidly as possible. This first phase of wound healing is nonmitotic in nature and consists entirely of cell migration. After a delay of many hours, the second phase of healing, marked by active mitosis, begins and serves to restore cellular density.1"4 Failure of an epithelial defect to heal may result in infection, enzymatic breakdown and permanent damage to the deeper structures. Many drugs have been investigated for the purpose of pharmacologically accelerating corneal epithelial healing. Epidermal growth factor (EGF) has been reported to increase corneal epithelial wound closure both in vivo and in vitro5"9 and to also stimulate corneal epithelial cell proliferation and DNA synthesis.8 Although EGF is considered primarily a mitoFrom the Departments of "Ophthalmology and fPathology, the University of Michigan Medical School, Ann Arbor, Michigan. Supported by grants from the Michigan Eye Bank and Transplantation Center (HKS), Fight for Sight, Inc. (HKS), and the American Cancer Society (grant PDT-324) (JV). Submitted for publication: November 1, 1988; accepted February 23, 1989. Reprint requests: H. Kaz Soong, MD, W. K. Kellogg Eye Center, 1000 Wall Street, Ann Arbor, MI 48105. •
genie agent, it also stimulates production of extracellular matrix components.10 In epidermal cells (keratinocytes), EGF not only increases mitosis, but also appears to directly stimulate cell motility.'' Although it has been commonly assumed that EGF may directly stimulate cell motility in corneal epithelial cells as well,6'7'912 no studies have as yet specifically addressed and proven this. We initially used two different assay methods (Boyden chamber1314 and agarose drop14"16) to study the effects of EGF on the motility of individual corneal epithelial cells in tissue culture. The advantage of both assay methods is that the motility of individual cells could be measured independently of mitotic influences. In addition, we used a third experimental method using organ-cultured corneas with epithelial wounds. In this method, the effects of EGF on corneal epithelial wound closure rates were studied in the presence and absence of colchicine. Colchicine, applied to the organ culture system in order to inhibit mitosis,1718 eliminates any acceleration of wound closure related to the mitogenic effects of EGF. Thus, in a multilayered cellular environment devoid of any background cellular proliferation, it is possible to determine if EGF is capable of directly stimulating corneal epithelial cell motility. The organ culture model
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EGF AND CORNEAL EPITHELIUM / Soong er ol
permits population migration behavior to be studied, while the Boyden chamber and agarose drop models permits individual cell migration properties to be studied. Materials and Methods Cell Cultures Adult male Sprague-Dawley rats and New Zealand albino rabbits were killed with an overdose of intraperitoneal (rat) or intravenous (rabbit) ketamine. Maintenance and handling of the animals were performed in accordance with NIH guidelines and the ARVO Resolution on the Use of Animals in Research. Full-thickness sheets of corneal epithelium were removed using neutral bacterial protease (Dispase II, Sigma Chemical Co., St. Louis, MO).19 The sheets and fragments of epithelium were explanted onto plastic tissue culture dishes and the cells were allowed to grow for 1 week into a monolayer at 37°C and 5% CO2 in denned minimum essential medium (DMEM) (Eagle's minimum essential medium with non-essential amino acids, L-glutamine, penicillin, streptomycin and amphotericin B) with 10% fetal calf serum and with or without 10 ng/ml EGF (Sigma). Transformed cells from a human oral (epithelial) squamous cell carcinoma line (SCC-1) in tissue culture were obtained from Dr. Thomas Carey (Department of Otolaryngology, University of Michigan Medical School, Ann Arbor, MI). Organ Cultures
Thirty adult male Sprague-Dawley rats (59 eyes) were killed with an overdose of intraperitoneal ketamine. A 4 mm diameter, central trephine mark was made on the surface of each cornea and the epithelium within was gently removed using a blunt, rounded-edge razor blade (cutting edge smoothly dulled on a fine whetstone). Wounded corneoscleral buttons were excised and mounted endothelialrsidedown onto dome-shaped paraffin platforms inside culture wells. The corneas were cultured for 21 hr at 37°C and 5% CO2 in DMEM without serum, and with or without drugs (colchicine and/or EGF). Drugs
For the Boyden chamber assay, EGF was added to the tissue culture medium at concentrations of 10 or 50 ng/ml. For the agarose drop assay, EGF was added to the medium at concentrations of 5, 50 or 100 ng/ml. The controls were free of EGF. The organ culture study was organized into two parts. The first part consisted of a comparison of epithelial wound healing rates between three popula-
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tions: Group 1) eleven corneas cultured in the presence of mouse submaxillary gland EGF (Sigma Chemicals, St. Louis, MO), alone, at a concentration of 50 ng/ml; Group 2) seventeen corneas cultured with both EGF (50 ng/ml) and colchicine (40 Mg/ml) (Sigma Chemicals); and Group 3) ten corneas (serving as controls for Groups 1 and 2) cultured in drugfree medium. The second part compared wound healing rates between ten corneas cultured in drugfree medium and 11 corneas cultured in colchicine (40 Mg/ml) alone. Assay of Motility in Modified Boyden Chamber
Modified blind-well Boyden chambers were used to study random cellular migration based on the method described by Romualdez and Ward.13 The chambers consist of two compartments partitioned by 12 /tm pore nitrocellulose filters (Schleicher and Schuell, Inc., Keene, NH). Top and bottom compartments each received 200 (x\ of serum-free DMEM. In some chambers, EGF (either 10 or 50 ng/ml) was added only to the bottom compartment, while in other chambers, EGF (50 ng/ml) was added to both the upper and lower compartments. For the controls, no EGF was added to. either compartment. Cells to be tested were seeded into the top compartment at concentrations of 3 X 105 (rat) and 5 X 105 (rabbit) cells per chamber. In order to provide a comparative positive control for migration, oral squamous carcinoma cells (SCC-1), which are known to increase random migration in response to EGF, were used in concurrent experiments with corneal epithelial cells. All chambers were incubated at 37°C in 5% CO2 for 18 hr. The filters were removed and stained with hematoxylin, dehydrated in propanol, cleared with xylene and mounted onto glass slides. Cell migration was quantitatively read by light microscopy as the number of cells per high-power field (X400) that migrated into the pores of the filter. Readings were performed in triplicate and compared statistically with the student t-test. Assay of Motility with Agarose Drop Explant Method
The agarose drop explant method of assaying random cellular migration was a modification of the methods described by Carpenter15 and by Harrington and Stastny.16 Cells to be assayed were trypsined, rinsed, and centrifuged into a pellet. The cells were resuspended at a concentration of 1 X 107 cells/ml in DMEM supplemented with 10% fetal calf serum and 0.2% agarose (Seaplaque Agarose, Marine Colloids, Rockville, ME). Agarose-cell droplets of 5 fi\ size were delivered by sterile micropipets and placed in the
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / August 1989
Table 1. Effects of EGF on cell migration in the Boyden chamber assay EGF concentration in compartments (ng/ml) Cells Rat
Rabbit
SCC-1
Number of migrated cells per high-power field (±SEM) at filter depth of 10 nm
Top
Bottom
0 50 0 0
0 50 50 10
4± 1 2± 1 3± 1
0 50 0 0
0 50 50 10
2± 2 1± 1 3± 2 4± 2
0 50 10
0 50 10
38 ± 11 128 ±25 74 ± 15
5± 2
Boyden Chamber Studies
Wound Studies (Organ Culture System)
At the end of the 21 hr organ culture incubation period, the cultured corneas were stained with RichTable 2. Effects of EGF on rat corneal epithelial cell migration, compared with SCC-1 positive controls, in agarose assay (data expressed as number of cells migrated outside agarose droplet) Number of migrated cells (±SEM) Concentration ofEGF (ng/ml)
18 h
48h
Rat
0 100 50 5
