Promotion of Corneal Epithelial Wound Healing by a Tetrapeptide (SSSR) Derived from IGF-1

Abstract

purpose. A prior study showed that a tetrapeptide (FGLM-amide) derived from the carboxyl terminus of substance P (SP) and a 12-residue peptide corresponding to the C domain of insulin-like growth factor (IGF)-1 mimic the synergistic effect of the full-length molecules on corneal epithelial wound healing. To develop an effective treatment for persistent corneal epithelial defects, the current study was conducted to investigate the minimal sequence within the C domain of IGF-1 that is required for such synergism with SP or FGLM-amide.

methods. The effects of IGF-1–derived peptides on corneal epithelial migration were evaluated with a rabbit corneal organ-culture system.

results. A tetrapeptide (SSSR; Ser³³-Ser-Ser-Arg) derived from the C domain of IGF-1 was sufficient for the synergistic promotion with FGLM-amide both of corneal epithelial migration in vitro and of wound closure in vivo. The activity of the SSSR peptide was sequence specific and its potency was similar to that of IGF-1. The SSSR peptide by itself also promoted corneal epithelial migration in vitro at higher concentrations. It was devoid, however, of both the mitogenic action of IGF-1 and the ability of the full-length molecule to induce neovascularization.

conclusions. The SSSR sequence mediates the synergistic effect of IGF-1 with SP on corneal epithelial wound healing. Clinical application of the SSSR peptide would be expected to be free of potentially deleterious side effects associated with treatment with full-length IGF-1. Local administration of the SSSR tetrapeptide, alone or in combination with FGLM-amide, is thus a potential new strategy for the treatment of nonhealing epithelial wounds.

Epithelia provide an essential barrier that protects organs and tissues from the external environment and contributes to maintenance of the internal environment of the body. Although epithelial defects in general heal relatively quickly, the persistence of such defects under certain conditions, such as those that exist in individuals with diabetes mellitus or peripheral neuropathy, can have serious pathologic consequences. The persistence of epithelial defects of the cornea often results in the development of corneal ulcer, which can lead to corneal perforation and loss of vision. There is currently no consistently effective mode of treatment for persistent corneal epithelial defects that is free of potentially adverse consequences. 

Damage to the corneal epithelium is followed by three phases of epithelial wound healing. Immediately after injury, the remaining epithelial cells begin to migrate to cover the area of the defect. After the defect is covered, contact inhibition results in the termination of epithelial cell migration and the epithelial cells enter a proliferative phase. Finally, the newly generated cells differentiate to form the stratified structure of the corneal epithelium. ¹ Epithelial migration is thus an important first step in corneal epithelial wound healing. To develop an effective treatment for persistent corneal epithelial defects, we have established an organ-culture system for the rabbit cornea that allows quantitative evaluation of the effects of test agents on epithelial migration. ² With this system, we have previously shown that several biological factors, including epidermal growth factor, ³ fibronectin, ² interleukin-6, ⁴ and the combination of insulin-like growth factor (IGF)-1 and substance P (SP) ⁵ promote epithelial migration in vitro. Verification of the relevance of this in vitro system was provided by the observation that the combination of IGF-1 and SP also facilitates closure of rabbit corneal epithelial wounds in vivo. ^{6 7} We have also shown that administration of eye drops containing both IGF-1 and SP is effective for the treatment of persistent corneal epithelial defects in individuals with neurotrophic keratopathy. ^{8 9 10}  

Both in vivo and in vitro studies have shown that SP exerts its synergistic effect with IGF-1 on corneal epithelial migration through interaction with the neurokinin receptor NK-1. ¹¹ The carboxyl-terminal four amino acids of SP (Phe-Gly-Leu-Met-amide, or FGLM-amide) are sufficient for this effect. ¹² The synergistic effect of IGF-1 with SP on epithelial migration is mimicked by IGF-2 but not by insulin. ¹³ IGFs and insulin share many structural similarities in their A and B domains. ¹⁴ However, the C domain of IGFs does not exhibit sequence homology to the C peptide of proinsulin, which is not retained in the mature insulin molecule. We have recently shown that the C domain of IGF-1 or -2 is responsible for the synergistic facilitation with SP of corneal epithelial migration. ¹⁵ This action of a peptide corresponding to the C domain of IGF-1 was independent of known IGF and insulin receptors, however. Furthermore, we found that the C-domain peptide by itself was able to promote epithelial migration, with SP functioning as a modulator by sensitizing epithelial cells to the action of the C domain. ¹⁶  

We have now examined the minimal sequence within the C domain of IGF-1 that is required for the synergistic facilitation with SP or FGLM-amide of corneal epithelial migration. We found that a tetrapeptide, SSSR (Ser³³-Ser-Ser-Arg), derived from the C domain is sufficient for this effect in vitro and in vivo. Furthermore, like the C-domain peptide, the SSSR tetrapeptide by itself was able to promote epithelial migration at high concentrations. The tetrapeptide was devoid of mitogenic action and did not induce neovascularization, both of which are potentially unfavorable side effects of treatment with the full-length IGF-1 molecule. The SSSR peptide is thus a potential new therapeutic agent for the treatment of persistent corneal epithelial defects. 

Materials and Methods

Recombinant human IGF-1 was obtained from BD Biosciences (Bedford, MA). Synthetic peptides derived from IGF-1, including SSSR, RSSS, SRSS, and SSRS, as well as FGLM-amide, were synthesized by the Peptide Institute (Osaka, Japan). Ethylene-vinyl-acetate (EVA) copolymer and methylene chloride were from Acros Organics (Geel, Belgium) and Wako (Osaka, Japan), respectively. 

Preparation of GST Fusion Proteins

Both strands of cDNAs encoding the Gly³⁰-Thr⁴¹ or Gly³²-Pro³⁹ peptides of human IGF-1, or alanine-scanning mutants of the latter peptide, were synthesized and cloned into the pGEX-3X vector (GE Healthcare, Piscataway, NJ). The resultant constructs were confirmed by DNA sequencing and introduced into bacteria for synthesis of the encoded glutathione S-transferase (GST) fusion proteins. The recombinant proteins were purified from bacterial cell lysates by chromatography on a glutathione-Sepharose column (GE Healthcare). 

Cell Culture

A human corneal epithelial cell line that had been transformed with a simian virus 40–adenovirus recombinant vector ¹⁷ was cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) until the cells had achieved ∼90% confluence in 100-mm culture dishes. Human corneal fibroblasts were prepared from donor tissue remaining after corneal transplantation surgery and were cultured as described previously. ¹⁸ The donor was a 65-year-old white man, and the cornea was obtained from Northwest Lions Eye Bank (Seattle, WA) and used in accordance with the tenets of the Declaration of Helsinki. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated FBS until they had achieved ∼90% confluence in 100-mm culture dishes, and they were subjected to experiments in the third to fifth passage. 

Animals

Albino Japanese rabbits (body mass, 2–3 kg) were obtained from Kitayama Laboratories (Kyoto, Japan) and KBT Oriental (Saga, Japan). Their care and treatment conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Rabbit Corneal Epithelial Migration Assay

The rabbit corneal epithelial migration assay was performed as described previously. ^{2 15} Rabbits were killed with an overdose of pentobarbital sodium injected intravenously, and both eyes were enucleated. The sclerocorneal rim was cut, and the cornea was excised and washed several times with sterile phosphate-buffered saline (PBS). Six tissue blocks were cut from each cornea with a razor blade, and each corneal block was placed with the epithelial side down in a well of a 24-well tissue culture plate containing medium-199 and test agents. After incubation for 24 hours at 37°C under a humidified atmosphere of 5% CO₂ in air, 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, the sections 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. 

Assay of Cell Proliferation

Human corneal epithelial cells or fibroblasts were cultured for 24 hours at 37°C in 24-well plates at a density of 1 × 10⁵/mL in DMEM supplemented with 0.2% FBS or with 0.1% bovine serum albumin, respectively. They were then cultured for an additional 24 or 20 hours, respectively, in corresponding culture medium containing test agent. After addition of [³H]thymidine (11 kBq; GE Healthcare), the cells were incubated for 1 hour, the medium was then removed, and 0.5 mL of 5% trichloroacetic acid was added to each well. The plate was incubated for 20 minutes at 4°C, after which the trichloroacetic acid was removed and each well was washed with 0.5 mL of H₂O. After the addition of 0.2 mL of 1 M NaOH to each well, the plate was incubated for 20 minutes at 37°C, 0.22 mL of 1 M HCl was added to each well, and the radioactivity present in the neutralized extract was measured with a scintillation counter. 

Assay of Neovascularization with the Corneal Pocket Model

EVA pellets were prepared as described previously. ¹⁹ In brief, an EVA copolymer was dissolved in methylene chloride at a concentration of 8% (wt/vol), and 5 μL of the solution was dried in a sterile plastic dish at room temperature. IGF-1 or SSSR peptide (10 μg) in PBS was then dropped onto the EVA film and allowed to dry at room temperature, after which another 5 μL of the EVA solution was layered on top of the dried residue. The resultant slow-release copolymer film was rolled up for use as an implant. Albino Japanese rabbits were anesthetized with an intramuscular injection of ketamine hydrochloride (50–80 mg/kg of body mass), and an incision was made in each cornea at a distance of 3 mm from the limbus. A microspatula was then inserted into the corneal stroma through each opening, and a cylindrical pocket was created. An implant was inserted into the pocket, and the wound was left unsutured. The extent of any resulting corneal neovascularization was monitored every 2 days. After 14 days, the cornea was photographed, and the area of neovascularization was measured with a computer-assisted digitizer. 

Assay of Rabbit Corneal Epithelial Wound Healing

Rabbits were anesthetized with an intramuscular injection of ketamine and xylazine as well as with oxybuprocaine eye drops, and the corneal epithelium of each eye was wounded with n-heptanol as described previously. ²⁰ Four animals were randomly assigned to each of four treatment groups and were treated with eye drops containing either 100 nM SSSR peptide, 1 mM FGLM-amide plus 100 nM IGF-1, 1 mM FGLM-amide plus 100 nM SSSR peptide, or PBS vehicle alone (control). Each eye received one drop of the respective solution immediately after n-heptanol treatment and again 2, 4, 6, 8, 10, 24, 26, 28, 30, 32, and 34 hours later. The epithelial defects were stained with one drop of 2% fluorescein and photographed immediately after wounding as well as 6, 12, 18, 24, 30, 36, and 48 hours later. The area of each epithelial defect was measured on the photographs with a computer-assisted digitizer. 

Statistical Analysis

Data are presented as the mean ± SE or SD. Statistical analysis was performed with the unpaired Student’s t-test for comparison of two groups or with the Dunnett or the Tukey-Kramer multiple comparison test for comparison of three or more groups. P < 0.05 was considered statistically significant. 

Results

Identification of IGF-1 Residues Required for Promotion of Corneal Epithelial Migration

We previously showed that a peptide corresponding to the C domain of human IGF-1 (Gly³⁰ to Thr⁴¹) mimics the synergistic effect of the full-length growth factor with SP or the SP-derived peptide FGLM-amide on corneal epithelial migration. ¹⁵ To delineate the region of the C domain of IGF-1 responsible for this effect, we first prepared an eight-residue peptide comprising Gly³² to Pro³⁹. In the presence of FGLM-amide, this peptide exhibited a synergistic effect on epithelial migration in our organ-culture system for the rabbit cornea similar to that of IGF-1 or the C-domain peptide (Fig. 1) . We next prepared a series of GST fusion proteins of the Gly³²-Pro³⁹ peptide and various mutants thereof in which residues were individually replaced by alanine (Fig. 2A) . Whereas GST alone had no effect on corneal epithelial migration in the absence or presence of FGLM-amide, the GST fusion protein of the Gly³²-Pro³⁹ peptide promoted epithelial migration in the presence of FGLM-amide by an extent similar to that apparent with the nonfused peptide (Figs. 1 2B) . Alanine-scanning mutagenesis revealed that substitution of Ser³³, Ser³⁴, Ser³⁵, or Arg³⁶, but not that of Gly³² or Arg³⁷, abolished the synergistic facilitation of corneal epithelial migration by the IGF-1–derived peptide (Fig. 2B) . These results indicated that the four-amino-acid sequence SSSR in the C domain of IGF-1 is essential for the synergistic effect with SP or FGLM-amide on corneal epithelial migration. Indeed, a synthetic peptide consisting of this sequence mimicked the synergistic effect of IGF-1 with FGLM-amide on this process (Fig. 3) . 

Characterization of the Effect of the SSSR Peptide on Corneal Epithelial Migration

We examined the specificity of the effect of the SSSR peptide on corneal epithelial migration in the presence of FGLM-amide. We synthesized three peptides (RSSS, SRSS, and SSRS) in which the SSSR sequence was scrambled. Each of these three peptides failed to facilitate corneal epithelial migration in the presence of FGLM-amide (Fig. 3) , indicating that the action of the SSSR peptide is sequence specific. The concentration-response relation for the SSSR peptide in the presence of FGLM-amide was also similar to that for IGF-1 (Fig. 4A) , with the median effective concentrations being 0.146 and 0.348 nM, respectively. The SSSR peptide was thus as potent as was IGF-1 in the synergistic promotion with FGLM-amide of corneal epithelial migration. As we previously showed for the C-domain peptide, ¹⁶ the SSSR peptide alone facilitated corneal epithelial migration, but only at concentrations higher than those effective in the presence of SP or FGLM-amide (Fig. 4B) . The median effective concentration of the SSSR peptide alone was 1.46 μM, which is similar to the value previously obtained for the C-domain peptide in the absence of SP or FGLM-amide. ¹⁶  

Effects of the SSSR Peptide on Cell Proliferation and Neovascularization

We next investigated whether the SSSR peptide possesses any other biological properties, such as the ability to stimulate the proliferation of corneal cells or to induce neovascularization. The potential mitogenic effect of the peptide was assessed by measurement of the incorporation of [³H]thymidine into DNA of cultured human corneal epithelial cells or human corneal fibroblasts. Whereas IGF-1 induced a concentration-dependent increase in the proliferation of both cell types, the SSSR peptide had no significant effect on cell proliferation at concentrations up to 100 nM (Fig. 5) . We further examined whether the SSSR peptide exerts a mitogenic action in the presence of FGLM-amide. Whereas IGF-1 at 100 nM induced a significant increase in the incorporation of [³H]thymidine in corneal epithelial cells in the absence or presence of FGLM-amide, the SSSR peptide at the same concentration had no effect on cell proliferation in the absence or presence of the SP-derived peptide (Fig. 6) . 

The induction of neovascularization by IGF-1 is an unfavorable activity from the standpoint of corneal wound healing. We therefore examined whether the SSSR peptide induces neovascularization in rabbits. Pellets containing the same weight (10 μg) of IGF-1 (1.3 nM) or the SSSR peptide (23 nM) were inserted into the corneal stroma of three rabbits. Two of the three corneas exposed to IGF-1 developed marked neovascularization, whereas none of the three corneas exposed to the SSSR peptide exhibited neovascularization (Fig. 7) . Quantitation of the area of neovascularization revealed mean (±SE) values of 4.23 ± 2.5 and 0 mm² (n = 3) for the IGF-1 and SSSR groups, respectively. No induction of neovascularization was observed with pellets containing PBS as a negative control. 

Promotion of Corneal Wound Healing In Vivo by the SSSR Peptide

Finally, we examined whether the SSSR peptide was able to facilitate the healing of rabbit corneal epithelial wounds in vivo. As shown previously, ¹² administration of eye drops containing 100 nM IGF-1 and 1 mM FGLM-amide significantly promoted epithelial wound closure in the rabbit cornea (Fig. 8) , compared with that apparent in control eyes treated with PBS. Whereas administration of eye drops containing the SSSR peptide alone at a concentration of 100 nM had no significant effect on corneal epithelial wound closure, treatment with drops containing both 100 nM SSSR and 1 mM FGLM-amide facilitated wound closure to an extent similar to that observed with the combination of IGF-1 and FGLM-amide (Fig. 8) . These results indicate that the SSSR tetrapeptide is able to substitute for IGF-1 in the synergistic facilitation with FGLM-amide of corneal epithelial wound closure in vivo. 

Discussion

We previously showed that the combination of IGF-1 and either SP or the SP-derived peptide FGLM-amide effectively promotes both corneal epithelial migration in vitro ⁵ and the closure of corneal epithelial wounds in vivo both in animals ¹² and in humans. ⁸ We have now demonstrated that the SSSR peptide possesses the same activity as full-length IGF-1 in the synergistic facilitation with FGLM-amide of corneal epithelial migration in vitro and corneal epithelial wound closure in vivo. Furthermore, at higher concentrations, the SSSR peptide alone promoted corneal epithelial migration in vitro. The activity of the SSSR tetrapeptide was sequence specific, and its potency was similar to that of IGF-1 in the presence of FGLM-amide. In contrast to IGF-1, however, the tetrapeptide was devoid of mitogenic action and of the ability to induce neovascularization. The SSSR peptide would thus appear to be a better choice than full-length IGF-1 for the treatment of corneal epithelial defects, in that it would be expected to be free of potentially deleterious side effects such as the induction of hyperplasia and neovascularization. 

We previously demonstrated that a mutant IGF-1 protein in which Ser³⁴ of the C domain was replaced by alanine, IGF-1(S34A), did not act synergistically with FGLM-amide to promote corneal epithelial migration. ¹⁵ We have now shown that the SS³⁴SR sequence of IGF-1 is essential for such synergism. IGF-2 reproduces the effect of IGF-1 with SP on corneal epithelial migration. ¹³ We also previously showed that the C domain of IGF-2 possesses the same activity in this regard as does the corresponding domain of IGF-1. ¹⁵ The C domains of IGF-1 and -2 consist of 12 and 8 amino acids, respectively. Although the SSSR sequence of IGF-1 is not present in the C domain of IGF-2 (SRVSRRSR), the two C domains may share a common functional determinant, the identity of which remains to be determined. 

IGF-1 stimulates the proliferation of various cell types. ²¹ Indeed, we recently showed that IGF-1 stimulates the proliferation of human corneal epithelial cells in culture. ²² Whereas the IGF-1(S34A) mutant retains the ability of the wild-type protein to stimulate cell proliferation through interaction with the IGF type 1 receptor, ¹⁵ we have now shown that the SSSR peptide did not stimulate the proliferation of corneal epithelial cells or corneal fibroblasts. No biological activity has been attributed to the C domain of IGF-1, although this domain modulates the interaction of the A and B domains of IGF-1 with the IGF type 1 receptor. ^{23 24 25} The present study also showed that, whereas IGF-1 induced neovascularization in the rabbit cornea, consistent with previous observations, ²⁶ the SSSR peptide did not exhibit such an effect. This biological activity of IGF-1 is also mediated by IGF type 1 receptors. ^{21 27} The effect of the SSSR peptide on corneal epithelial migration thus appears to be independent of known IGF receptors, given that the peptide is devoid both of the mitogenic action of wild-type IGF-1 and of its ability to induce neovascularization. Our findings thus implicate a novel mechanism in the promotion of epithelial migration by IGF-1. It is possible that this action of the SSSR peptide is mediated by an unidentified receptor. Alternatively, the peptide may penetrate the cell membrane and thereby affect intracellular signaling directly. Further investigations of the mechanism of action of the SSSR peptide in the promotion of corneal epithelial migration are thus necessary. 

Cell migration plays an important role during the initial phase of corneal epithelial wound healing. ^{1 28} After the epithelial defect is covered by a monolayer of migrating epithelial cells, cell proliferation contributes to restoration of the original thickness of the epithelium. Both fibronectin and epidermal growth factor stimulate corneal epithelial wound healing. Whereas fibronectin stimulates only the migration of corneal epithelial cells, epidermal growth factor stimulates both cell migration and proliferation. ³ The effects of these two agents on corneal epithelial wound healing thus appear to be mediated by different mechanisms. Given the possible adverse consequences of stimulation of cell proliferation, agents that stimulate only the first phase of corneal epithelial wound healing (the migration of epithelial cells) may be preferable for the treatment of persistent corneal epithelial defects. We have now shown that, by shortening the IGF-1 molecule to the SSSR sequence of the C domain, we were able to eliminate the mitogenic activity of intact IGF-1 but retain the stimulatory effect on epithelial migration. 

FGLM-amide is able to substitute for SP in the promotion, together with IGF-1 of corneal epithelial wound healing. ¹² FGLM-amide lacks the ability of SP ²⁹ to induce contraction of the pupil (miosis). We have now delineated the region of IGF-1 necessary for promotion of corneal epithelial wound healing as the SSSR tetrapeptide. These two short peptides (FGLM-amide and SSSR) would be expected to have little antigenicity, which, together with their lack of other biological activities of the parent molecules, would be advantageous in the clinical setting. Furthermore, we demonstrated the effectiveness of the SSSR peptide in the absence of FGLM-amide in the promotion of corneal epithelial migration in vitro. Treatment with this single peptide may thus further eliminate potentially adverse biological activities of FGLM-amide mediated by the NK-1 receptor. Treatment with the SSSR peptide alone may prove suitable for persistent corneal epithelial defects caused by viral infection or by eye surgery, whereas the combination of SSSR and FGLM-amide may be more effective for the treatment of persistent defects caused by neurotrophic keratopathy, given that the level of SP is reduced in the cornea of individuals with this condition. 

The corneal epithelium shares several characteristics with the skin. Both tissues are of ectodermal origin and possess sensory nerve fibers that contain SP. The two tissues may thus share a common mechanism of wound healing, in which case the combination of FGLM-amide and the SSSR peptide may prove effective for the treatment of skin injuries. Indeed, our preliminary results indicate that this combination of peptides promotes the healing of skin wounds. Our results thus provide the basis for development of a new strategy for the treatment of corneal and skin injuries by the application of two short peptides associated with minimal adverse effects. 

 Supported by grants from the Japan Society for the Promotion of Science (JSPS) and from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MI, TNi).

 Submitted for publication September 11, 2005; revised March 8, 2006; accepted May 23, 2006.

 Disclosure: N. Yamada, None; R. Yanai, None; K. Kawamoto, None; T. Nagano, Santen Pharmaceutical Co. Ltd. (E); M. Nakamura, Santen Pharmaceutical Co. Ltd. (E); M. Inui (P); T. Nishida (P)

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Effects of synthetic peptides derived from IGF-1 on corneal epithelial migration. Rabbit corneal blocks were incubated for 24 hours with IGF-1, the C-domain peptide (Gly³⁰-Thr⁴¹), or the Gly³²-Pro³⁹ peptide at a concentration of 1 nM in the absence or presence of 20 μM FGLM-amide. The extent of epithelial migration was then determined. Data are expressed as a percentage of the value for corneal blocks incubated with test agent in the absence of FGLM-amide and are the mean ± SE of six determinations. **P < 0.01, ***P < 0.001 versus corresponding corneal blocks incubated without FGLM-amide.

Figure 2.

 

Minimal sequence of the C domain of IGF-1 required for synergistic facilitation with FGLM-amide of corneal epithelial migration. (A) Schematic representation of a series of GST fusion proteins containing the C domain (Gly³⁰-Thr⁴¹) of wild-type IGF-1, the Gly³²-Pro³⁹ peptide, or X→Ala mutants of the latter peptide. (B) Rabbit corneal blocks were incubated for 24 hours with GST or the indicated GST fusion proteins, each at a concentration of 1 nM, in the absence or presence of 20 μM FGLM-amide, after which the length of the path of epithelial migration was determined. Data are expressed as a percentage of the value for corneal blocks incubated with test agent in the absence of FGLM-amide and are means ± SE of values from six to eight determinations. *P < 0.05, **P < 0.01 versus corresponding corneal blocks incubated without FGLM-amide.

Figure 3.

 

Effects of SSSR and control peptides with FGLM-amide on corneal epithelial migration. Rabbit corneal blocks were incubated for 24 hours with SSSR, RSSS, SRSS, or SSRS peptides, each at a concentration of 1 nM, in the absence or presence of 20 μM FGLM amide. The length of the path of epithelial migration was then measured. Data are expressed as a percentage of the value for corneal blocks incubated with test peptide in the absence of FGLM-amide and are means ± SE of six determinations. ***P < 0.001 versus corresponding corneal blocks incubated without FGLM-amide.

Figure 4.

 

Concentration-response relations for the effects of the SSSR peptide on corneal epithelial migration in the presence or absence of FGLM-amide. Rabbit corneal blocks were incubated for 24 hours with the indicated concentrations of IGF-1 or SSSR peptide in the presence (A) or absence (B) of 20 μM FGLM-amide, after which the extent of epithelial migration was determined. Data are expressed as percentage facilitation of epithelial migration relative to control incubations (corneal blocks incubated alone) and are means ± SE of six determinations. *P < 0.05, **P < 0.01 compared with respective control.

Figure 5.

 

Lack of effect of the SSSR peptide on cell proliferation. Incorporation of [³H]thymidine into human corneal epithelial cells (A) or human corneal fibroblasts (B) was measured after incubation with the indicated concentrations of IGF-1 or the SSSR peptide. Data are means ± SE of results of three independent experiments. ***P < 0.001 versus cells incubated without test agent.

Figure 6.

 

Lack of effect of the SSSR peptide on cell proliferation in the presence of FGLM-amide. Incorporation of [³H]thymidine into human corneal epithelial cells was measured after incubation with 100 nM IGF-1 or 100 nM SSSR peptide in the absence or presence of 20 μM FGLM-amide. Data are expressed as a percentage of the value for cells incubated in the absence of test agents and are the mean ± SE of results of three independent experiments. ***P < 0.001 versus cells incubated without test agents.

Figure 7.

 

Lack of effect of the SSSR peptide on neovascularization in the rabbit corneal pocket model. Pellets containing the SSSR peptide (10 μg) or IGF-1 (10 μg) were implanted into a pocket introduced into the rabbit cornea (arrowheads), and the cornea was photographed 14 days later. Arrow: neovascularization induced by IGF-1.

Figure 8.

 

Effect of the SSSR peptide on rabbit corneal epithelial wound closure in vivo. After removal of the corneal epithelium by exposure to n-heptanol, eyes were treated with eye drops containing PBS vehicle (control), 100 nM SSSR peptide, 100 nM IGF-1 plus 1 mM FGLM-amide, or 100 nM SSSR peptide plus 1 mM FGLM-amide. Representative photographs of the cornea 24 hours after removal of the epithelium (A) and the area of the epithelial defects at various times after n-heptanol treatment (B) are shown. Data in (B) are expressed as a percentage of the corresponding value for PBS-treated eyes and are the mean ± SD of results for eight eyes. *P < 0.05, **P < 0.01, ***P < 0.001 versus the corresponding value for PBS-treated eyes.