March 2007
Volume 48, Issue 3
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Cornea  |   March 2007
Stimulation of Corneal Epithelial Migration by a Synthetic Peptide (PHSRN) Corresponding to the Second Cell-Binding Site of Fibronectin
Author Affiliations
  • Kazuhiro Kimura
    From the Departments of Ocular Pathophysiology and
  • Atsushi Hattori
    Nitten Pharmaceutical Co., Ltd., Nagoya, Japan; and the
  • Yumiko Usui
    Nitten Pharmaceutical Co., Ltd., Nagoya, Japan; and the
  • Kayo Kitazawa
    Nitten Pharmaceutical Co., Ltd., Nagoya, Japan; and the
  • Masumi Naganuma
    Nitten Pharmaceutical Co., Ltd., Nagoya, Japan; and the
  • Koji Kawamoto
    Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan;
  • Shinichiro Teranishi
    Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan;
  • Motoyoshi Nomizu
    Department of Clinical Biochemistry, Tokyo University of Pharmacy and Life Science, Tokyo, Japan.
  • Teruo Nishida
    From the Departments of Ocular Pathophysiology and
    Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan;
Investigative Ophthalmology & Visual Science March 2007, Vol.48, 1110-1118. doi:10.1167/iovs.06-0704
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      Kazuhiro Kimura, Atsushi Hattori, Yumiko Usui, Kayo Kitazawa, Masumi Naganuma, Koji Kawamoto, Shinichiro Teranishi, Motoyoshi Nomizu, Teruo Nishida; Stimulation of Corneal Epithelial Migration by a Synthetic Peptide (PHSRN) Corresponding to the Second Cell-Binding Site of Fibronectin. Invest. Ophthalmol. Vis. Sci. 2007;48(3):1110-1118. doi: 10.1167/iovs.06-0704.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Fibronectin plays an important role in the migration of corneal epithelial cells in vivo. The Arg-Gly-Asp (RGD) sequence in the principal cell binding domain of fibronectin mediates the interaction of fibronectin with integrins, whereas the Pro-His-Ser-Arg-Asn (PHSRN) sequence of fibronectin is thought to modulate this interaction. The authors examined the effects of a PHSRN peptide on corneal epithelial migration in vitro and in vivo.

methods. Epithelial migration in vitro was examined with the rabbit cornea in organ culture. The motility and phenotype of simian virus 40–transformed human corneal epithelial (HCE) cells were evaluated by time-lapse and immunofluorescence microscopy, respectively. Tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin was examined by immunoprecipitation and immunoblot analysis. The healing of rabbit corneal epithelial wounds induced by 1-heptanol was evaluated by fluorescein staining.

results. The PHSRN peptide stimulated corneal epithelial migration in organ culture in a concentration-dependent manner, and it increased HCE cell motility in vitro. The peptide induced the accumulation of F-actin and the formation of focal adhesions at the leading edge of HCE cells. It also upregulated the tyrosine phosphorylation of FAK and paxillin in HCE cells, but it did not affect HCE cell proliferation or attachment to a fibronectin matrix. Administration of the PHSRN peptide in eye drops promoted corneal epithelial wound closure in vivo in a dose-dependent manner. None of these effects of the PHSRN peptide were induced by a control NRSHP peptide.

conclusions. The PHSRN peptide mimics many of the effects of fibronectin on corneal epithelial cells and may prove suitable as a substitute for fibronectin in the treatment of persistent corneal epithelial defects.

The healing of corneal epithelial wounds is important for maintaining corneal transparency. Damage to the corneal epithelium induces migration of the remaining epithelial cells to cover the area of the defect. Thus, the motility of corneal epithelial cells is key to the initiation of wound healing. We and others 1 2 have shown that fibronectin, an adhesive extracellular glycoprotein, appears at the exposed surface of the stroma at corneal epithelial wound sites. Epithelial cells migrate over this provisional matrix of fibronectin and upregulate their expression of integrin chains that form the cell surface receptors for fibronectin. 3 The fibronectin–integrin system thus plays a central role in corneal epithelial wound healing. 4 Adding exogenous fibronectin stimulates corneal epithelial migration in an organ culture system of the rabbit cornea. 5 Furthermore, a fibronectin matrix promotes the attachment of corneal epithelial cells in culture, and this effect is inhibited by the addition of an RGD (Arg-Gly-Asp) peptide corresponding to the principal cell-binding domain of fibronectin (Gly-Arg-Gly-Asp-Ser-Pro [GRGDSP]). 6 7 8 Exogenous fibronectin does not affect the proliferation of cultured corneal epithelial cells. 9 10 11 12 13 The administration of fibronectin eye drops also facilitates corneal epithelial wound closure in experimental animals 14 15 16 17 and has proven effective clinically for the treatment of persistent epithelial defects of the cornea. 5 18 19 20 21  
Fibronectin is a dimer of two almost identical polypeptides. It consists of several structural domains, including three types of repeat, that mediate the various biological functions of the protein. 22 23 The principal cell-binding domain of fibronectin has been localized to the 10th type III repeat, with the RGD sequence most important for the binding of fibronectin to integrins, including the α5β1 and αIIIbβ3 complexes. In addition to the RGD sequence, the PHSRN (Pro-His-Ser-Arg-Asn) sequence, located in the ninth type III repeat, has been implicated as a second cell-binding site of fibronectin. This sequence is thought to promote cell spreading on fibronectin mediated by the RGD sequence. 24 It is thus considered a modulatory site that facilitates the binding of fibronectin to integrins. 25 26 27 28 Administration of a PHSRN peptide has been shown to facilitate the healing of skin wounds by promoting the migration of keratinocytes. 29  
We investigated whether the PHSRN peptide might also promote the healing of corneal epithelial wounds. We examined the possible effects of the peptide on epithelial migration in an organ culture system of the rabbit cornea, on the motility and proliferation of cultured human corneal epithelial cells, and on the phosphorylation of focal adhesion proteins in these cells. Finally, we examined the effect of administration of the PHSRN peptide on corneal epithelial wound closure in rabbits. 
Methods
Animals
Japanese albino rabbits (each weighing approximately 3 kg) were obtained from Chubu Kagaku Sizai (Nagoya, Japan). All animal procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Materials
The following materials were used for this study: sodium pentobarbital (Tokyo Kasei Kogyo, Tokyo, Japan); ketamine hydrochloride (Wako, Osaka, Japan); xylazine hydrochloride (Bayer, Tokyo, Japan); oxybuprocaine hydrochloride (Neovenol Ophthalmic Solution, Nitten Pharmaceutical, Nagoya, Japan); 1-heptanol and sodium fluorescein (uranine; Wako); human fibronectin (hFN), Dulbecco modified Eagle medium–nutrient mixture F12 (DMEM-F12), medium-199, Ca2+- and Mg2+-free phosphate-buffered saline (PBS), fetal bovine serum (FBS), trypsin-EDTA, and gentamicin (Invitrogen-Gibco, Grand Island, NY); bovine serum albumin (BSA), recombinant bovine insulin, cholera toxin, recombinant human epidermal growth factor (rhEGF), and a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO); bovine fibronectin (bFN; Roche, Basel, Switzerland). Plastic culture dishes (100-mm and 96-well; Corning, Corning, NY); 35-mm glass-bottomed culture dishes (Iwaki, Tokyo, Japan); enzyme-linked immunosorbent assay (ELISA) kit for detection of bromodeoxyuridine (BrdU; Roche, Indianapolis, IN); protein G–Sepharose, a gelatin-coupled Sepharose 4B column, horseradish peroxidase–conjugated rabbit antibodies to mouse immunoglobulin G (IgG), and ECL Plus detection reagents (Amersham Biosciences, Little Chalfont, UK); Cellufine resin column (Chisso, Tokyo, Japan); mouse monoclonal antibodies to phosphotyrosine and to focal adhesion kinase (UBI, Temecula, CA); mouse monoclonal antibodies to paxillin (BD Biosciences, San Jose, CA); TOTO-3, calcein-AM, Alexa Fluor 488–labeled goat antibodies to mouse IgG and rhodamine-phalloidin (Invitrogen, Carlsbad, CA); mouse monoclonal antibodies to fibronectin (Takara Bio, Shiga, Japan); acetyl-PHSRN-amide and acetyl-NRSHP-amide peptides (Peptide Institute, Osaka, Japan); and GRGDSP and GRGESP (Takara, Kyoto, Japan). 
Assay of Rabbit Corneal Epithelial Migration
Rabbit corneal epithelial migration in vitro was measured as described previously. 30 31 Rabbits were killed with an overdose of sodium pentobarbital injected intravenously, and both eyes of each rabbit were enucleated. The sclerocorneal rim was cut, and the cornea was excised and washed several times with sterile PBS. Six tissue blocks (each approximately 2 × 4 mm) were cut from each cornea with a razor blade, and each corneal block was placed with the epithelial side up in a well of a 24-well tissue culture plate containing medium-199 in the absence (control) or presence of various concentrations of PHSRN or NRSHP peptides. rhEGF and hFN at 100 nM were used as positive controls. After incubation for 24 hours at 37°C, the blocks were fixed overnight at 4°C with a mixture of glacial acetic acid and absolute ethanol (5:95, vol/vol). They were then dehydrated by exposure to a graded series of ethanol solutions, immersed in chloroform, and embedded in paraffin. Four thin (7-μm) sections were cut at 200-μm intervals from each block and, after the removal of paraffin, were stained with hematoxylin-eosin. The sections were examined with a light microscope and photographed, and the length of the path of corneal epithelial migration down both sides of each section (toward the endothelial side) was measured on the photographs with a computer-assisted digitizer. The eight measurements for each corneal block were averaged. 
Cells and Cell Culture
Simian virus 40–immortalized human corneal epithelial (HCE) cells, originally established and characterized by Kaoru Araki-Sasaki, 32 were provided (Riken Biosource Center, Tsukuba, Japan). They were passaged in supplemented hormonal epithelial medium (SHEM), composed of DMEM-F12 supplemented with 15% heat-inactivated FBS, bovine insulin (5 μg/mL), cholera toxin (0.1 μg/mL), rhEGF (10 ng/mL), and gentamicin (40 μg/mL). For experiments, HCE cells were cultured for 24 hours in unsupplemented DMEM-F12, isolated by treatment with trypsin-EDTA, suspended in the same medium, and plated at a density of 2 × 104 cells per 35-mm dish, 2 × 106 cells per 100-mm dish, or 1 × 103 cells or 2 × 104 per well of a 96-well plate. 
Assay of Cell Motility
HCE cells were plated on 35-mm glass-bottomed culture dishes in unsupplemented DMEM-F12 and were cultured for 6 hours, after which the medium was changed to DMEM-F12, with or without 2 μM PHSRN or NRSHP peptides, and the cells were placed in a humidified incubator containing 5% CO2 at 37°C. One to four cells not in contact with other cells in each field were then monitored with the use of a fluorescence inverted microscope (Axioscope; Carl Zeiss, Hallbergmoos, Germany). Phase-contrast images were collected every 6 minutes for 6 hours with a charge-coupled device camera (Axiocam; Carl Zeiss). Cell motility was quantified by tracking the positions of nuclei with the use of software (Move-tr/2D; Library, Tokyo, Japan). 
Immunofluorescence Microscopy
HCE cells were plated on 35-mm glass-bottomed culture dishes in unsupplemented DMEM-F12, cultured for 6 hours, and incubated for 90 minutes in DMEM-F12 containing the PHSRN or NRSHP peptides (2 μM). The cells were then fixed with 3.7% formalin for 15 minutes at 37°C, washed with PBS, and incubated for 1 hour at room temperature first with 1% BSA in PBS and then with antibodies to phosphotyrosine (1:200 dilution in PBS containing 1% BSA). After washing with PBS, the cells were incubated for 1 hour with Alexa Fluor 488–conjugated goat secondary antibodies (1:1000 dilution), rhodamine-phalloidin (1:200 dilution), and TOTO-3 (1:1000 dilution) in PBS containing 1% BSA. They were then washed; those not in contact with other cells were examined with a laser confocal microscope (LSM5; Carl Zeiss). 
Immunoprecipitation and Immunoblot Analysis
HCE cells were cultured in 100-mm plastic culture dishes in unsupplemented DMEM-F12 for 24 hours, washed twice with PBS, and incubated in DMEM-F12 containing the PHSRN or NRSHP peptides (2 μM) for 90 minutes. After two washes with PBS, the cells were lysed in 300 μL solution containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 5 mM NaF, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM Na3VO4, and 1% protease inhibitor cocktail. The lysates were centrifuged at 15,000g for 10 minutes at 4°C, and portions of each supernatant (100 μg protein) were incubated for 16 hours at 4°C in a final volume of 200 μL with antibodies to FAK or to paxillin (1:100 dilutions) and 20 μL protein G-Sepharose beads. The beads were isolated by centrifugation and washed twice with cell lysis solution, and the bound proteins were fractionated by SDS-PAGE on an 8.5% gel and then transferred to a nitrocellulose membrane. After incubation for 16 hours at 4°C with blocking buffer (20 mM Tris-HCl [pH 7.4], 5% skim milk, 0.1% Tween 20), the membrane was exposed for 16 hours at 4°C to antibodies to phosphotyrosine (1:1000 dilution in blocking buffer), washed with 20 mM Tris-HCl (pH 7.4) containing 0.1% Tween 20, incubated for 1 hour at room temperature with horseradish peroxidase–conjugated secondary antibodies (1:1000 dilution in blocking buffer), and washed again. Immune complexes were detected with ECL Plus (Amersham) reagents. 
Assay of Cell Adhesion
The wells of a 96-well plate were coated with bFN (0.5 μg/mL) for 1 hour at room temperature and then exposed for 1 hour to 1% BSA in PBS. HCE cells plated on a 100-mm dish were cultured for 2 hours in DMEM-F12 containing 5 μM calcein-AM, washed twice with PBS, and isolated by exposure to trypsin-EDTA. Cells were incubated for 30 minutes at 37°C with DMEM-F12 containing various concentrations of GRGDSP, GRGESP, PHSRN, or NRSHP peptides, and the incubation mixtures were then transferred to the wells of the bFN-coated plate (2 × 104 cells per well). After incubation of the plate for 1 hour at 37°C, the incubation mixtures were removed, the wells were washed twice with PBS, and the attached cells were quantified by measurement of calcein fluorescence with a fluorescence plate reader (CytoFluorII; PerSeptive Biosystems, Foster City, CA) at excitation and emission wavelengths of 490 and 515 nm, respectively. 
Assay of Cell Proliferation
HCE cells plated in 96-well dishes in unsupplemented DMEM-F12 were cultured for 24 hours, after which the medium was replaced with DMEM-F12 containing various concentrations of PHSRN or NRSHP peptides (or 10% FBS as a positive control). After incubation of the cells for 48 hours, cell proliferation was measured with the use of an assay kit. In brief, BrdU solution was added to the culture medium in each well, and the cells were incubated for an additional 2 hours. The medium was then removed from each well, and the cells were processed for colorimetric detection of incorporated BrdU with ELISA reagents. The absorbance of each well was measured at 370 nm with a fluorescence plate reader. 
Purification of Rabbit Plasma Fibronectin
Rabbit fibronectin (rFN) was purified from the plasma fraction of 10 mL blood collected into a syringe containing sodium citrate (final concentration, 10%). The plasma was applied to a column of gelatin–Sepharose 4B that had been equilibrated with PBS containing 10% sodium citrate. The column was washed with the equilibration solution, and rFN was then eluted with 1 M arginine. The eluted fraction was applied to a resin column (Cellufine; Chisso Corp.) to remove arginine; elution was performed with PBS and monitored by measurement of absorbance at 280 nm. Fractions containing rFN were concentrated by centrifugation through a dialysis membrane. Purity of the rFN preparation was confirmed by Coomassie brilliant blue staining and immunoblot analysis with antibodies to fibronectin (Fig. 1)
Assay of Rabbit Corneal Epithelial Wound Healing
Rabbits (total, 65; 10 eyes per treatment group) were anesthetized with intramuscular injection of ketamine (40 mg/kg body mass) and xylazine (4 mg/kg), and each eye was subjected to topical anesthesia by instillation of oxybuprocaine hydrochloride; the animals recovered from the general anesthesia after 1 to 2 hours. A corneal epithelial wound was introduced into each eye of the anesthetized animals as described previously. 33 34 In brief, a filter paper disc (6 mm in diameter) soaked with 1-heptanol was positioned in the center of each corneal surface and left in place for 1 minute. The disc was then removed, and the damaged corneal epithelium was washed thoroughly with sterile saline. PHSRN or NRSHP peptides (0.02–2000 μM) in PBS (30 μL) were applied topically to each eye immediately and at 6, 12, 18, 24, 30, 36, 42, 48, and 54 hours after wounding; rhEGF and rFN were used as positive controls at concentrations of 5 and 1 μM, respectively, and PBS was used as a negative control. The area of the corneal epithelial defect was stained with one drop of 1% sodium fluorescein and immediately photographed at 6-hour intervals up to 54 hours after wounding. The stained area was measured by computerized planimetry with NIH Image (Image J software, version 1.62f; available by ftp at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). The rate of epithelial wound closure was calculated for each eye as the slope of the best-fit straight line determined by least-squares regression analysis for the linear period (6–32 hours) of healing, during which the area of the defect decreased by approximately 80%. 
Statistical Analysis
Quantitative data are presented as mean ± SEM or SD. Differences among groups were evaluated by analysis of variance (ANOVA) followed by Dunnett multiple comparison test. P < 0.05 was considered statistically significant. 
Results
We first examined the effect of the PHSRN peptide on epithelial migration in an organ culture system of the rabbit cornea (Fig. 2) . The length of the path of migration of the corneal epithelium over the cut surface of the stroma during incubation for 24 hours was increased by the addition of hFN (100 nM) or rhEGF (100 nM), both of which were used as positive controls. 13 30 The PHSRN peptide also increased the extent of corneal epithelial migration in a concentration-dependent manner; the effect was significant at concentrations of 200 and 2000 nM (Figs. 2A 2C) . The peptide NRSHP, whose sequence was reversed compared with that of PHSRN, had no effect on corneal epithelial migration at any of the concentrations tested (Figs. 2B 2D) , indicating that the effect of the PHSRN peptide was sequence specific. 
We next examined whether the PHSRN peptide affected the motility of HCE cells in culture. Cells cultured with the PHSRN peptide (2 μM) manifested pronounced motility accompanied by the formation of membrane ruffling at the cell periphery (Fig. 3A) . In contrast, cells cultured with the NRSHP peptide (2 μM) or in medium alone did not move substantially or manifest dynamic morphologic changes. Time-lapse analysis of the position of cell nuclei during incubation of the cells for 6 hours revealed that the distance moved by nuclei in the presence of the PHSRN peptide was increased approximately fourfold compared with that apparent in the presence of the NRSHP peptide or in the absence of test agent (Fig. 3B)
To examine further the effect of the PHSRN peptide on HCE morphology, we exposed the cells for 90 minutes to PHSRN or NRSHP peptides at a concentration of 2 μM. The cells were then fixed and stained for F-actin, tyrosine-phosphorylated proteins, and nuclei. The addition of the PHSRN peptide induced the formation of a thick rim of actin filaments in a dense meshwork at the leading edge of HCE cells (Fig. 4A) . Costaining with antibodies to phosphotyrosine revealed numerous small dotlike structures, presumably corresponding to focal adhesions, associated with the bundles of F-actin at the cell periphery. Cells treated with the NRSHP peptide exhibited only a thin rim of F-actin staining and only a few small dotlike structures reactive with the antibodies to phosphotyrosine at the cell periphery. To examine the effect of the PHSRN peptide on tyrosine phosphorylation of the focal adhesion proteins FAK and paxillin, we incubated HCE cells with the PHSRN or NRSHP peptides (2 μM) for 90 minutes and then subjected them to immunoprecipitation and immunoblot analysis. Tyrosine phosphorylation of FAK and paxillin was upregulated by the PHSRN peptide compared with that apparent for cells incubated with the NRSHP peptide (Fig. 4B)
We next examined whether the PHSRN peptide affected HCE cell adhesion to a fibronectin matrix. Neither the PHSRN peptide nor the NRSHP peptide, at a concentration of 1 to 1000 μM, had a significant effect on cell adhesion to fibronectin (Fig. 5A) . In contrast, the GRGDSP peptide, but not the control GRGESP peptide, inhibited the adhesion of HCE cells to fibronectin in a concentration-dependent manner. Incubation of HCE cells for 48 hours with the PHSRN or NRSHP peptides at concentrations of 0.2 to 200 μM also had no effect of cell proliferation (Fig. 5B)
Finally, we examined the effect of eye drops containing the PHSRN peptide on corneal epithelial wound closure in vivo. The corneal epithelium of rabbits was debrided with 1-heptanol, and eye drops containing the PHSRN or NRSHP peptides at 200 μM were then administered for 54 hours at intervals of 6 hours. As positive controls, eye drops containing 1 μM rFN or 5 μM rhEGF were administered. Fluorescein staining revealed that the epithelial defect was completely resurfaced by 36 hours in most eyes treated with eye drops containing the PHSRN peptide, rhEGF, or rFN, whereas complete resurfacing required 54 hours in eyes treated with PBS alone or with the NRSHP peptide (Fig. 6A) . Furthermore, whereas eyes treated with PBS or the NRSHP peptide manifested a delay of 6 hours before the commencement of a linear phase of epithelial wound healing, those treated with the PHSRN peptide, rFN, or rhEGF exhibited an immediate onset of wound healing (Fig. 6B) . Determination of the rate of healing between 6 and 32 hours after the initiation of treatment revealed that the PHSRN peptide stimulated wound healing in a dose-dependent manner; the effect was statistically significant at concentrations of 60, 200, and 2000 μM (Fig. 7) . Administration of the NRSHP peptide at concentrations up to 2000 μM did not affect the healing rate compared with that for eyes treated with PBS
Discussion
We have shown that the PHSRN peptide, which is based on the second cell-binding site of fibronectin located in the ninth type III repeat, stimulated corneal epithelial migration in vitro and corneal epithelial wound healing in vivo with an efficacy similar to that of fibronectin itself. This peptide also induced the tyrosine phosphorylation of FAK and paxillin, the accumulation of F-actin, and the formation of focal adhesions at the leading edge of HCE cells. Furthermore, the PHSRN peptide increased the motility of individual HCE cells, but it did not affect their proliferation or attachment to a fibronectin matrix. These various effects of the PHSRN peptide appeared to be sequence specific given that they were not mimicked by a peptide with the reverse sequence (NRSHP). 
Previous studies have shown that peptides derived from extracellular matrix proteins, such as collagen, fibronectin, and elastin, exhibit biological activities such as stimulation of cell chemotaxis, migration, and adhesion, 29 35 36 but the receptors and intracellular signaling pathways responsible for mediating these effects have remained unclear. We have now shown that the PHSRN peptide upregulated the tyrosine phosphorylation of FAK and paxillin in HCE cells. Phosphorylation and activation of FAK and paxillin have been implicated in the spreading and migration of epithelial cells, endothelial cells, and fibroblasts. 37 38 39 40 41 With regard to whether the effects of the PHSRN peptide on FAK and paxillin phosphorylation are mediated by integrins, the PHSRN sequence has been shown to modulate the binding of the RGD sequence of fibronectin to integrins. 22 42 43 Several studies have also suggested that the PHSRN peptide may interact with α5β1 integrin. 25 27 44 45 Moreover, crystallographic analysis has revealed that the RGD and PHSRN sequences of fibronectin are likely positioned close to integrins at the cell surface. 46 In contrast, molecular electron microscopy has revealed that, although the RGD sequence in the 10th type III repeat of fibronectin appears to interact with α5β1 integrin, the PHSRN sequence in the ninth such repeat does not. 47 Protein engineering, thermodynamics analysis, and nuclear magnetic resonance imaging have indicated that the ninth type III repeat of fibronectin is relatively unstable compared with the 10th such repeat. 48 49 We have shown that the PHSRN peptide did not have a significant effect on HCE cell adhesion to fibronectin. It is difficult to reconcile these various observations with regard to the role of the PHSRN sequence at the molecular level in the interaction of fibronectin with integrins, but it is possible that the PHSRN peptide binds to a cell surface protein other than integrins. 
Our present results showing the facilitation of corneal epithelial wound healing by the PHSRN peptide are consistent with the previously observed effects of the peptide on keratinocytes. 29 Application of the PHSRN peptide in powder form to a round skin wound made by trephination in mice was found to promote healing at an effective dose of 4 μg. The maximally effective concentration of PHSRN peptide we used in eye drops was 200 μM; given that we applied 30 μL eye drops at each treatment, we added 3.9 μg of the PHSRN peptide to each eye at each administration. Moreover, the PHSRN peptide at 200 nM increased rabbit corneal epithelial migration in organ culture to an extent similar to that observed with 100 nM hFN. There was no apparent difference in corneal epithelial migration and attachment during wound healing between eyes treated with the PHSRN peptide and those treated with rFN. 
PHSRN peptide–induced tyrosine phosphorylation of FAK and paxillin detected in HCE cells was likely responsible for the associated increase in cell motility. FAK and paxillin activation triggers characteristic changes in cell shape, actin cytoskeleton organization, and focal adhesions that are mediated by members of the Rho family of proteins. Activation of Rho thus induces the assembly of parallel arrays of actin stress fibers and promotes the formation of well-defined focal adhesions, 50 whereas activation of Rac elicits the formation of lamellipodia and actin recruitment to membrane ruffles and that of Cdc42 results in the formation of filopodia and membrane microspikes. 51 Rac and Cdc42 also induce the assembly of small focal adhesions termed focal contacts. 52 We previously showed that fibronectin activates Rac1 and induces the formation of membrane ruffles and focal adhesions, resulting in increases in cell adhesion and motility, in HCE cells. 53 Fibronectin has also been shown to activate Rho and Cdc42 and thereby to regulate cell adhesion and migration. 54 55 We have now shown that the PHSRN peptide triggered the formation of F-actin bundles and focal adhesions at the periphery of HCE cells. However, in contrast to fibronectin, the PHSRN peptide increased cell motility without affecting cell adhesion. These observations suggest that the PHSRN peptide activates Rho family proteins in HCE cells but that the balance of its effects on these proteins may differ from that of the effects of fibronectin. 
Since we first reported the clinical efficacy of fibronectin eye drops in the treatment of persistent epithelial defects of the cornea, 16 fibronectin for such treatment has not become commercially available, primarily because fibronectin is isolated from blood and is therefore susceptible to contamination by blood-borne infectious agents. We continue to prepare autologous fibronectin from the serum of each patient for this purpose. 18 Producing a recombinant form of the protein is also difficult because the glycation of fibronectin, a large glycoprotein, is essential for its biological functions. Our results now suggest that the use of eye drops containing a synthetic peptide (PHSRN) based on the second cell-binding site of fibronectin is a promising approach for the treatment of persistent epithelial defects of the cornea. 
 
Figure 1.
 
Purification of fibronectin from rabbit plasma. Purified rFN (8 ng) was subjected to SDS-PAGE on a 7.5% gel and either stained with Coomassie brilliant blue (CBB) or further subjected to immunoblot analysis (IB) with antibodies to fibronectin.
Figure 1.
 
Purification of fibronectin from rabbit plasma. Purified rFN (8 ng) was subjected to SDS-PAGE on a 7.5% gel and either stained with Coomassie brilliant blue (CBB) or further subjected to immunoblot analysis (IB) with antibodies to fibronectin.
Figure 2.
 
Effects of the PHSRN and NRSHP peptides, hFN, and rhEGF on epithelial migration in an organ culture system of the rabbit cornea. Corneal blocks were incubated for 24 hours in the absence (control) or presence of hFN (100 nM), rhEGF (100 nM), or various concentrations (2–2000 nM) of the PHSRN (A, C) or NRSHP (B, D) peptides, after which sections of the tissue were stained with hematoxylin-eosin. Representative sections are shown (C, D). Arrows and arrowheads indicate the initial and final positions, respectively, of the edge of the epithelium. Scale bar, 200 μm. Path length of epithelial migration was determined, and quantitative data (mean ± SEM) from four independent experiments are shown (A, B). **P < 0.01 compared with control group (ANOVA followed by Dunnett multiple comparison test).
Figure 2.
 
Effects of the PHSRN and NRSHP peptides, hFN, and rhEGF on epithelial migration in an organ culture system of the rabbit cornea. Corneal blocks were incubated for 24 hours in the absence (control) or presence of hFN (100 nM), rhEGF (100 nM), or various concentrations (2–2000 nM) of the PHSRN (A, C) or NRSHP (B, D) peptides, after which sections of the tissue were stained with hematoxylin-eosin. Representative sections are shown (C, D). Arrows and arrowheads indicate the initial and final positions, respectively, of the edge of the epithelium. Scale bar, 200 μm. Path length of epithelial migration was determined, and quantitative data (mean ± SEM) from four independent experiments are shown (A, B). **P < 0.01 compared with control group (ANOVA followed by Dunnett multiple comparison test).
Figure 3.
 
Effects of the PHSRN and NRSHP peptides on HCE cell motility. Cells were incubated in the absence (control) or presence of PHSRN or NRSHP peptide at a concentration of 2 μM, and cell movement was monitored by time-lapse video microscopy for 6 hours at 6-minute intervals. Representative images are shown (A), as is quantitation of the distance moved by individual cell nuclei (B). Quantitative data are mean ± SD of nuclei in a representative experiment. *P < 0.05 compared with control (ANOVA followed by Dunnett test). Similar results were obtained in three independent experiments.
Figure 3.
 
Effects of the PHSRN and NRSHP peptides on HCE cell motility. Cells were incubated in the absence (control) or presence of PHSRN or NRSHP peptide at a concentration of 2 μM, and cell movement was monitored by time-lapse video microscopy for 6 hours at 6-minute intervals. Representative images are shown (A), as is quantitation of the distance moved by individual cell nuclei (B). Quantitative data are mean ± SD of nuclei in a representative experiment. *P < 0.05 compared with control (ANOVA followed by Dunnett test). Similar results were obtained in three independent experiments.
Figure 4.
 
Effects of the PHSRN peptide on the actin cytoskeleton and focal adhesions in HCE cells. (A) Cells were exposed to PHSRN or NRSHP peptide (2 μM) for 90 minutes, fixed, and stained with antibodies to phosphotyrosine (green) to detect focal adhesions, with rhodamine-phalloidin (red) to detect actin filaments, and with TOTO-3 (blue) to detect nuclei. Scale bar, 10 μm. (B) Cells were incubated with PHSRN or NRSHP peptide (2 μM) for 90 minutes, lysed, and subjected to immunoprecipitation (IP) with antibodies to FAK or to paxillin. Resultant precipitates were then subjected to immunoblot analysis (IB) with antibodies to phosphotyrosine (pTyr) and with the antibodies used for immunoprecipitation.
Figure 4.
 
Effects of the PHSRN peptide on the actin cytoskeleton and focal adhesions in HCE cells. (A) Cells were exposed to PHSRN or NRSHP peptide (2 μM) for 90 minutes, fixed, and stained with antibodies to phosphotyrosine (green) to detect focal adhesions, with rhodamine-phalloidin (red) to detect actin filaments, and with TOTO-3 (blue) to detect nuclei. Scale bar, 10 μm. (B) Cells were incubated with PHSRN or NRSHP peptide (2 μM) for 90 minutes, lysed, and subjected to immunoprecipitation (IP) with antibodies to FAK or to paxillin. Resultant precipitates were then subjected to immunoblot analysis (IB) with antibodies to phosphotyrosine (pTyr) and with the antibodies used for immunoprecipitation.
Figure 5.
 
Lack of effect of the PHSRN peptide on HCE cell adhesion to a fibronectin matrix or on HCE cell proliferation. (A) Cells were labeled with calcein-AM and then incubated for 30 minutes at 37°C with various concentrations (1–1000 μM) of the synthetic peptides PHSRN, NRSHP, GRGDSP, and GRGESP. Incubation mixtures were then added to the wells of a 96-well plate coated with bFN (0.5 μg/mL), and the plate was incubated for 1 hour at 37°C, after which the wells were washed and the number of attached cells was evaluated by measurement of calcein fluorescence. Data are mean ± SD of triplicates from an experiment that was repeated three times. (B) Cells were cultured for 48 hours in DMEM-F12 in the absence (negative control) or presence of various concentrations (0.2–200 μM) of PHSRN or NRSHP peptides or of 10% FBS (positive control). Cell proliferation was then evaluated by measurement of BrdU incorporation with ELISA. Data are mean ± SD of triplicates from an experiment that was repeated three times. *P < 0.05 compared with negative control (ANOVA followed by Dunnett test).
Figure 5.
 
Lack of effect of the PHSRN peptide on HCE cell adhesion to a fibronectin matrix or on HCE cell proliferation. (A) Cells were labeled with calcein-AM and then incubated for 30 minutes at 37°C with various concentrations (1–1000 μM) of the synthetic peptides PHSRN, NRSHP, GRGDSP, and GRGESP. Incubation mixtures were then added to the wells of a 96-well plate coated with bFN (0.5 μg/mL), and the plate was incubated for 1 hour at 37°C, after which the wells were washed and the number of attached cells was evaluated by measurement of calcein fluorescence. Data are mean ± SD of triplicates from an experiment that was repeated three times. (B) Cells were cultured for 48 hours in DMEM-F12 in the absence (negative control) or presence of various concentrations (0.2–200 μM) of PHSRN or NRSHP peptides or of 10% FBS (positive control). Cell proliferation was then evaluated by measurement of BrdU incorporation with ELISA. Data are mean ± SD of triplicates from an experiment that was repeated three times. *P < 0.05 compared with negative control (ANOVA followed by Dunnett test).
Figure 6.
 
Effect of the PHSRN peptide on corneal epithelial wound closure in vivo. (A) Rabbit corneal epithelial defects induced by 1-heptanol were stained with fluorescein at the indicated times after the onset of treatment with eye drops containing PBS alone, the PHSRN or NRSHP peptides (200 μM), rFN (1 μM), or rhEGF (5 μM). (B) Time course of the healing of corneal epithelial defects induced and treated as in (A). The healed area was expressed as a percentage of the initial wound area. Data are mean ± SEM of values for 10 eyes. **P < 0.01 for the PHSRN peptide, rhEGF, or rFN groups compared with the PBS group. †P < 0.05 for the rhEGF group compared with the PBS group (ANOVA followed by Dunnett test).
Figure 6.
 
Effect of the PHSRN peptide on corneal epithelial wound closure in vivo. (A) Rabbit corneal epithelial defects induced by 1-heptanol were stained with fluorescein at the indicated times after the onset of treatment with eye drops containing PBS alone, the PHSRN or NRSHP peptides (200 μM), rFN (1 μM), or rhEGF (5 μM). (B) Time course of the healing of corneal epithelial defects induced and treated as in (A). The healed area was expressed as a percentage of the initial wound area. Data are mean ± SEM of values for 10 eyes. **P < 0.01 for the PHSRN peptide, rhEGF, or rFN groups compared with the PBS group. †P < 0.05 for the rhEGF group compared with the PBS group (ANOVA followed by Dunnett test).
Figure 7.
 
Effect of the PHSRN peptide on the healing rate of corneal epithelial wounds in vivo. Rabbit corneal epithelial defects induced by 1-heptanol were treated with eye drops containing PBS alone, PHSRN or NRSHP peptides (0.02–2000 μM), rFN (1 μM), or rhEGF (5 μM), and the healing rate of the epithelial defects was determined. Data are mean ± SEM of values for 10 eyes. *P < 0.05. **P < 0.01 compared with the PBS group (ANOVA followed by Dunnett test).
Figure 7.
 
Effect of the PHSRN peptide on the healing rate of corneal epithelial wounds in vivo. Rabbit corneal epithelial defects induced by 1-heptanol were treated with eye drops containing PBS alone, PHSRN or NRSHP peptides (0.02–2000 μM), rFN (1 μM), or rhEGF (5 μM), and the healing rate of the epithelial defects was determined. Data are mean ± SEM of values for 10 eyes. *P < 0.05. **P < 0.01 compared with the PBS group (ANOVA followed by Dunnett test).
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Figure 1.
 
Purification of fibronectin from rabbit plasma. Purified rFN (8 ng) was subjected to SDS-PAGE on a 7.5% gel and either stained with Coomassie brilliant blue (CBB) or further subjected to immunoblot analysis (IB) with antibodies to fibronectin.
Figure 1.
 
Purification of fibronectin from rabbit plasma. Purified rFN (8 ng) was subjected to SDS-PAGE on a 7.5% gel and either stained with Coomassie brilliant blue (CBB) or further subjected to immunoblot analysis (IB) with antibodies to fibronectin.
Figure 2.
 
Effects of the PHSRN and NRSHP peptides, hFN, and rhEGF on epithelial migration in an organ culture system of the rabbit cornea. Corneal blocks were incubated for 24 hours in the absence (control) or presence of hFN (100 nM), rhEGF (100 nM), or various concentrations (2–2000 nM) of the PHSRN (A, C) or NRSHP (B, D) peptides, after which sections of the tissue were stained with hematoxylin-eosin. Representative sections are shown (C, D). Arrows and arrowheads indicate the initial and final positions, respectively, of the edge of the epithelium. Scale bar, 200 μm. Path length of epithelial migration was determined, and quantitative data (mean ± SEM) from four independent experiments are shown (A, B). **P < 0.01 compared with control group (ANOVA followed by Dunnett multiple comparison test).
Figure 2.
 
Effects of the PHSRN and NRSHP peptides, hFN, and rhEGF on epithelial migration in an organ culture system of the rabbit cornea. Corneal blocks were incubated for 24 hours in the absence (control) or presence of hFN (100 nM), rhEGF (100 nM), or various concentrations (2–2000 nM) of the PHSRN (A, C) or NRSHP (B, D) peptides, after which sections of the tissue were stained with hematoxylin-eosin. Representative sections are shown (C, D). Arrows and arrowheads indicate the initial and final positions, respectively, of the edge of the epithelium. Scale bar, 200 μm. Path length of epithelial migration was determined, and quantitative data (mean ± SEM) from four independent experiments are shown (A, B). **P < 0.01 compared with control group (ANOVA followed by Dunnett multiple comparison test).
Figure 3.
 
Effects of the PHSRN and NRSHP peptides on HCE cell motility. Cells were incubated in the absence (control) or presence of PHSRN or NRSHP peptide at a concentration of 2 μM, and cell movement was monitored by time-lapse video microscopy for 6 hours at 6-minute intervals. Representative images are shown (A), as is quantitation of the distance moved by individual cell nuclei (B). Quantitative data are mean ± SD of nuclei in a representative experiment. *P < 0.05 compared with control (ANOVA followed by Dunnett test). Similar results were obtained in three independent experiments.
Figure 3.
 
Effects of the PHSRN and NRSHP peptides on HCE cell motility. Cells were incubated in the absence (control) or presence of PHSRN or NRSHP peptide at a concentration of 2 μM, and cell movement was monitored by time-lapse video microscopy for 6 hours at 6-minute intervals. Representative images are shown (A), as is quantitation of the distance moved by individual cell nuclei (B). Quantitative data are mean ± SD of nuclei in a representative experiment. *P < 0.05 compared with control (ANOVA followed by Dunnett test). Similar results were obtained in three independent experiments.
Figure 4.
 
Effects of the PHSRN peptide on the actin cytoskeleton and focal adhesions in HCE cells. (A) Cells were exposed to PHSRN or NRSHP peptide (2 μM) for 90 minutes, fixed, and stained with antibodies to phosphotyrosine (green) to detect focal adhesions, with rhodamine-phalloidin (red) to detect actin filaments, and with TOTO-3 (blue) to detect nuclei. Scale bar, 10 μm. (B) Cells were incubated with PHSRN or NRSHP peptide (2 μM) for 90 minutes, lysed, and subjected to immunoprecipitation (IP) with antibodies to FAK or to paxillin. Resultant precipitates were then subjected to immunoblot analysis (IB) with antibodies to phosphotyrosine (pTyr) and with the antibodies used for immunoprecipitation.
Figure 4.
 
Effects of the PHSRN peptide on the actin cytoskeleton and focal adhesions in HCE cells. (A) Cells were exposed to PHSRN or NRSHP peptide (2 μM) for 90 minutes, fixed, and stained with antibodies to phosphotyrosine (green) to detect focal adhesions, with rhodamine-phalloidin (red) to detect actin filaments, and with TOTO-3 (blue) to detect nuclei. Scale bar, 10 μm. (B) Cells were incubated with PHSRN or NRSHP peptide (2 μM) for 90 minutes, lysed, and subjected to immunoprecipitation (IP) with antibodies to FAK or to paxillin. Resultant precipitates were then subjected to immunoblot analysis (IB) with antibodies to phosphotyrosine (pTyr) and with the antibodies used for immunoprecipitation.
Figure 5.
 
Lack of effect of the PHSRN peptide on HCE cell adhesion to a fibronectin matrix or on HCE cell proliferation. (A) Cells were labeled with calcein-AM and then incubated for 30 minutes at 37°C with various concentrations (1–1000 μM) of the synthetic peptides PHSRN, NRSHP, GRGDSP, and GRGESP. Incubation mixtures were then added to the wells of a 96-well plate coated with bFN (0.5 μg/mL), and the plate was incubated for 1 hour at 37°C, after which the wells were washed and the number of attached cells was evaluated by measurement of calcein fluorescence. Data are mean ± SD of triplicates from an experiment that was repeated three times. (B) Cells were cultured for 48 hours in DMEM-F12 in the absence (negative control) or presence of various concentrations (0.2–200 μM) of PHSRN or NRSHP peptides or of 10% FBS (positive control). Cell proliferation was then evaluated by measurement of BrdU incorporation with ELISA. Data are mean ± SD of triplicates from an experiment that was repeated three times. *P < 0.05 compared with negative control (ANOVA followed by Dunnett test).
Figure 5.
 
Lack of effect of the PHSRN peptide on HCE cell adhesion to a fibronectin matrix or on HCE cell proliferation. (A) Cells were labeled with calcein-AM and then incubated for 30 minutes at 37°C with various concentrations (1–1000 μM) of the synthetic peptides PHSRN, NRSHP, GRGDSP, and GRGESP. Incubation mixtures were then added to the wells of a 96-well plate coated with bFN (0.5 μg/mL), and the plate was incubated for 1 hour at 37°C, after which the wells were washed and the number of attached cells was evaluated by measurement of calcein fluorescence. Data are mean ± SD of triplicates from an experiment that was repeated three times. (B) Cells were cultured for 48 hours in DMEM-F12 in the absence (negative control) or presence of various concentrations (0.2–200 μM) of PHSRN or NRSHP peptides or of 10% FBS (positive control). Cell proliferation was then evaluated by measurement of BrdU incorporation with ELISA. Data are mean ± SD of triplicates from an experiment that was repeated three times. *P < 0.05 compared with negative control (ANOVA followed by Dunnett test).
Figure 6.
 
Effect of the PHSRN peptide on corneal epithelial wound closure in vivo. (A) Rabbit corneal epithelial defects induced by 1-heptanol were stained with fluorescein at the indicated times after the onset of treatment with eye drops containing PBS alone, the PHSRN or NRSHP peptides (200 μM), rFN (1 μM), or rhEGF (5 μM). (B) Time course of the healing of corneal epithelial defects induced and treated as in (A). The healed area was expressed as a percentage of the initial wound area. Data are mean ± SEM of values for 10 eyes. **P < 0.01 for the PHSRN peptide, rhEGF, or rFN groups compared with the PBS group. †P < 0.05 for the rhEGF group compared with the PBS group (ANOVA followed by Dunnett test).
Figure 6.
 
Effect of the PHSRN peptide on corneal epithelial wound closure in vivo. (A) Rabbit corneal epithelial defects induced by 1-heptanol were stained with fluorescein at the indicated times after the onset of treatment with eye drops containing PBS alone, the PHSRN or NRSHP peptides (200 μM), rFN (1 μM), or rhEGF (5 μM). (B) Time course of the healing of corneal epithelial defects induced and treated as in (A). The healed area was expressed as a percentage of the initial wound area. Data are mean ± SEM of values for 10 eyes. **P < 0.01 for the PHSRN peptide, rhEGF, or rFN groups compared with the PBS group. †P < 0.05 for the rhEGF group compared with the PBS group (ANOVA followed by Dunnett test).
Figure 7.
 
Effect of the PHSRN peptide on the healing rate of corneal epithelial wounds in vivo. Rabbit corneal epithelial defects induced by 1-heptanol were treated with eye drops containing PBS alone, PHSRN or NRSHP peptides (0.02–2000 μM), rFN (1 μM), or rhEGF (5 μM), and the healing rate of the epithelial defects was determined. Data are mean ± SEM of values for 10 eyes. *P < 0.05. **P < 0.01 compared with the PBS group (ANOVA followed by Dunnett test).
Figure 7.
 
Effect of the PHSRN peptide on the healing rate of corneal epithelial wounds in vivo. Rabbit corneal epithelial defects induced by 1-heptanol were treated with eye drops containing PBS alone, PHSRN or NRSHP peptides (0.02–2000 μM), rFN (1 μM), or rhEGF (5 μM), and the healing rate of the epithelial defects was determined. Data are mean ± SEM of values for 10 eyes. *P < 0.05. **P < 0.01 compared with the PBS group (ANOVA followed by Dunnett test).
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