Promotion of corneal epithelial wound healing by a tetrapeptide (SSSR) derived from IGF-1

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; Ser(33)-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.     

Sensitizing effect of substance P on corneal epithelial migration induced by IGF-1, fibronectin, or interleukin-6

PURPOSE: Substance P (SP) is present in the sensory nerve fibers of the corneal epithelium. Various biological agents, including epidermal growth factor, fibronectin, interleukin-6, and the combination of SP and insulin-like growth factor (IGF)-1, promote the healing of corneal epithelial wounds. The role of SP in corneal epithelial migration was examined. METHODS: The effects of various agents on corneal epithelial migration were investigated with the rabbit cornea in an organ culture system. RESULTS: An SP-derived tetrapeptide, FGLM-amide, shifted the dose-response relations for the induction of corneal epithelial migration not only by an IGF-1-derived peptide (C-domain peptide) but also by fibronectin or interleukin-6 to lower concentrations. This action of SP was prevented by inhibitors of phospholipase C, of the inositol 1,4,5-trisphosphate receptor-mediated release of Ca(2+) from intracellular stores, and of Ca(2+)- and calmodulin-dependent protein kinase II (CaM-PK II). CONCLUSIONS: These results indicate that SP, acting at the neurokinin type 1 receptor, functions as an important modulator of corneal epithelial wound healing by activating CaM-PK II in epithelial cells and thereby sensitizing them to the induction of migration by various biological agents. They also provide important insight into a new strategy for the treatment of corneal wounds.     

Signaling regulation for synergistic effects of substance P and insulin-like growth factor-1 or epidermal growth factor on corneal epithelial migration

PURPOSE: In a previous report we showed that substance P (SP) and insulin-like growth factor-1 (IGF-1) or epidermal growth factor (EGF) synergistically stimulate corneal epithelial migration. In this study, we used an organ culture system of rabbit cornea to identify which signal transduction system affects corneal epithelial migration. METHODS: Rabbit corneal blocks were cultured in TC-199 culture medium containing various reagents for 24 hours. After the end of cultivation, the length of the path of epithelial migration was measured. RESULTS: Acting alone, protein kinase C (PKC) inhibitors, calphostin C and H-7, each reduced the length of epithelial migration. Tyrosine kinase (TK) inhibitors, genistein and herbimycin A, also acted individually to inhibit epithelial migration. The synergistic stimulatory effects of SP and IGF-1 on corneal epithelial migration were eliminated when PKC inhibitors or TK inhibitors were added. The synergistic effect of SP and EGF was eliminated by TK inhibitors, but only partly suppressed by PKC inhibitors. CONCLUSIONS: These results suggest that the synergistic effect of SP and EGF might require a TK pathway, and that the synergistic effect of SP and IGF-1 might require both PKC and TK pathways.     

Characterization of insulin-like growth factor-1 receptors in rabbit corneal epithelial cells

Substance P (SP) and insulin-like growth factor-1 (IGF-1) synergistically facilitate corneal epithelial wound healing in vitro and in vivo. This synergism is mediated through the NK-1 receptors for SP, and IGF-1 does not modulate the binding affinity of NK-1 receptors. To clarify the effect of SP on the binding characteristics of IGF-1 receptors, the binding affinity and number of binding sites for IGF-1 in rabbit corneal epithelial cells were studied using a binding assay for(125)I-IGF-1. The binding affinity and number of binding sites for IGF-1 were determined by Scatchard plot analysis. Cultured rabbit corneal epithelial cells bound specifically to IGF-1. For IGF-1 in corneal epithelial cells, the binding affinity was 4 n m and the number of binding sites was 1x10(5)binding sites cell(-1). Although IGF-2 and insulin also bind to IGF-1 receptors, their affinities were, respectively, eight- and 300-fold lower than that of IGF-1. IGF-1 and IGF-2 stimulated corneal epithelial migration in the presence of SP, but insulin did not. Pretreatment of the corneal epithelial cells with SP (2x10(-5)m) failed to change the binding affinity or number of binding sites for IGF-1. These results demonstrated that corneal epithelial cells possess specific receptors for IGF-1. The synergistic effect of SP and IGF-1 on corneal epithelial wound healing does not result from regulation at the receptor level.     

Persistent Epithelial Defects Due to Neurotrophic Keratopathy Treated with a Substance P-Derived Peptide and Insulin-Like Growth Factor 1

Purpose

The loss of corneal sensation results in the development of persistent corneal epithelial defects. The combination of a substance P-derived peptide, phenylalanine -glycine-leucine-methionine (FGLM)-amide, and insulin-like growth factor 1 (IGF-1) stimulates corneal epithelial migration in vitro and corneal epithelial wound closure in vivo. The clinical efficacy of eye drops containing FGLM-amide and IGF-1 for the treatment of persistent epithelial defects in individuals with neurotrophic keratopathy was examined in a prospective open study.

Methods

Eleven patients with persistent corneal epithelial defects associated with neurotrophic keratopathy were treated for up to 28 days by the administration of eye drops containing FGLM-amide and IGF-1. The course of epithelial healing was monitored by slit-lamp examination, and visual acuity was measured before and after treatment.

Results

Complete epithelial resurfacing was achieved in eight of the nine (89%) cases of persistent epithelial defects in the nine patients who received the full course of treatment. Epithelial defects had completely resurfaced in two of nine (22%) and five of nine (56%) cases within 1 and 2 weeks, respectively, after treatment initiation. No adverse effects of treatment were observed in any of the 11 patients.

Conclusions

Eye drops containing FGLM-amide and IGF-1 induced the rapid resurfacing of persistent epithelial defects in individuals with neurotrophic keratopathy.?Jpn J Ophthalmol 2007;51:442–447 © Japanese Ophthalmological Society 2007

     

Restoration of corneal epithelial barrier function and wound healing by substance P and IGF-1 in rats with capsaicin-induced neurotrophic keratopathy

PURPOSE: To investigate the effects of topical application of the combination of substance P (SP) and insulin-like growth factor (IGF)-1 on corneal epithelial barrier function and epithelial wound closure in rats with capsaicin-induced neurotrophic keratopathy. METHODS: Neonatal rats were injected subcutaneously with a single dose of capsaicin to induce neurotrophic keratopathy. Corneal epithelial barrier function was evaluated with an anterior fluorophotometer. Tear fluid secretion was measured by the Schirmer test. Corneal epithelial wound healing was determined by measurement of the size of the epithelial defect after debridement of the entire epithelium. The combination of SP (1 mM) and IGF-1 (1 micro g/mL) in phosphate-buffered saline was administered in eye drops six times daily. RESULTS: Corneal epithelial barrier function was impaired and corneal epithelial wound healing was delayed in rats injected with capsaicin. The application of eye drops containing the combination of SP and IGF-1 to capsaicin-injected rats resulted in a significant improvement in corneal epithelial barrier function compared with that apparent in capsaicin-injected animals that received eye drops containing vehicle alone. Such treatment with SP and IGF-1 also significantly increased the rate of corneal epithelial wound closure in capsaicin-injected animals. CONCLUSIONS: Topical application of the combination of SP and IGF-1 improved both corneal epithelial barrier function and epithelial wound healing in an animal model of neurotrophic keratopathy.     

Peptide therapies for ocular surface disturbances based on fibronectin–integrin interactions

The condition of the corneal epithelium is a critical determinant of corneal transparency and clear vision. The corneal epithelium serves as a barrier to protect the eye from external insults, with its smooth surface being essential for its optical properties. Disorders of the corneal epithelium include superficial punctate keratopathy, corneal erosion, and persistent epithelial defects (PEDs). The prompt resolution of these disorders is important for minimization of further damage to the cornea. Currently available treatment modalities for corneal epithelial disorders are based on protection of the ocular surface in order to allow natural healing to proceed. PEDs remain among the most difficult corneal conditions to treat, however. On the basis of characterization of the pathobiology of PEDs at the cell and molecular biological levels, we have strived to develop new modes of treatment for these defects. These treatments rely on two key concepts: provision of a substrate, such as the adhesive glycoprotein fibronectin, for the attachment and migration of corneal epithelial cells, and activation of these cells by biological agents such as the combination of substance P and insulin-like growth factor-1 (IGF-1). Central to both approaches is the role of the fibronectin–integrin system in corneal epithelial wound healing. Determination of the minimum amino acid sequences required for the promotion of corneal epithelial wound closure by fibronectin (PHSRN) and by substance P (FGLM-amide) plus IGF-1 (SSSR) has led to the development of peptide eyedrops for the treatment of PEDs that are free of adverse effects of the parent molecules.     

Neurotrophic mediators and corneal wound healing

Neurotrophic keratopathy is an ocular pathological condition that remains difficult to treat. The loss of trigeminal nerve function and corneal sensation that underlies this condition can lead to the development of various disorders of the cornea. Substance P, a sensory neurotransmitter produced by the trigeminal nerve, has been investigated for its effect on corneal epithelial wound healing. Substance P by itself has no direct effect on corneal epithelial migration, but it manifests a synergistic action with insulin-like growth factor-1 (IGF-1) in both epithelial migration in vitro and corneal wound healing in vivo. The minimal amino acid sequences of both substance P and IGF-1 that are required for such effects have been determined. With use of these minimal amino acid sequences, the potential adverse consequences of treatment with the full-length polypeptides may be avoided. The application of eye drops containing a substance P-derived peptide and IGF-1 has proved clinically effective for the treatment of patients with persistent epithelial defects of the cornea.     

Stimulation of corneal epithelial migration by a synthetic peptide (PHSRN) corresponding to the second cell-binding site of fibronectin

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 effect of substance P with insulin-like growth factor-1 on corneal epithelial cell proliferation]

PURPOSE: To elucidate the pathogenesis of neuroparalytic keratopathy, we examined the effect of substance P (SP) on corneal epithelial cell proliferation in comparison with that of insulin-like growth factor-1 (IGF-1) in vivo and in vitro. METHODS: In the in vivo study, the epithelium of rabbit cornea was removed mechanically and treated with eye drops containing SP, IGF-1, SP + IGF-1, or physiological saline as a control. Corneas were excised 48 hours after the removal of the epithelium, labeled with 3H-thymidine, and subjected to autoradiography. An in vitro study was also performed by culturing rabbit corneal epithelial cells in culture medium containing SP with or without IGF-1 and labeling cells with 3H-thymidine for a subsequent autoradiographical study. FINDINGS: In the in vivo study, SP in combination with IGF-1 enhanced the healing of rabbit corneal epithelial defect. 3H-thymidine autoradiography revealed that in both in vivo and in vitro studies, SP + IGF-1 stimulated corneal epithelial cell proliferation, but SP or IGF-1 alone did not. CONCLUSION: These results indicate that SP enhances the stimulatory effect of IGF-1 on corneal epithelial cell proliferation and accelerates corneal epithelial healing.     

Role of JNK-dependent serine phosphorylation of paxillin in migration of corneal epithelial cells during wound closure

PURPOSE: Migration of corneal epithelial cells plays an important role in healing of corneal epithelial wounds. The role of c-Jun NH(2)-terminal kinase (JNK), a member of the family of mitogen-activated protein kinases, in the intracellular signaling responsible for the migration of corneal epithelial cells during wound closure was examined. METHODS: Scratch wounds were introduced into cultured monolayers of simian virus 40-transformed human corneal epithelial (HCE) cells in the absence or presence of the JNK inhibitor SP600125. The phosphorylation and localization of JNK and paxillin during wound closure were examined by immunoblot and immunofluorescence analyses. The effects of a small interfering RNA (siRNA) specific for JNK and of a mutant form of paxillin on HCE cell migration were determined by transfection. RESULTS: SP600125 inhibited wound healing in a time- and concentration-dependent manner. Immunoblot analysis showed that wounding increased the phosphorylation of JNK and of paxillin on serine (Ser) 178 in a manner sensitive to SP600125. Immunofluorescence staining revealed that phosphorylated JNK colocalized with paxillin at focal adhesions formed by HCE cells at the wound margin and that SP600125 inhibited the formation of such adhesions. Expression of JNK siRNA or of a paxillin mutant in which Ser178 is replaced by alanine inhibited HCE cell migration during wound closure. CONCLUSIONS: JNK regulates HCE cell migration by modulating the phosphorylation of paxillin and the consequent formation of focal adhesions. A JNK-paxillin signaling pathway may thus play an important role in corneal epithelial wound healing in vivo.     

The role of fibronectin in corneal wound healing explored by a physician–scientist

For the past 30?years, I have worked as a physician-scientist in both the clinic and laboratory setting at a number of university medical schools. Encountering patients in the clinic for whom treatment was not available led me to the laboratory in an attempt to develop the appropriate treatment for future patients. The main focus of my translational research has been the role of fibronectin in corneal epithelial wound healing and the development of fibronectin eyedrops for the treatment of patients with persistent corneal epithelial defects. An extension of this research led to the development of eyedrops containing the synthetic peptide proline-histidine-serine-arginine-asparagine (PHSRN), which corresponds to the second cell-binding site of fibronectin. My clinical experience with fibronectin eyedrops also prompted me to examine the role of the sensory neurotransmitter substance P and insulin-like growth factor-1 (IGF-1) in corneal wound healing, leading to the development of eyedrops containing peptides derived from these agents (peptides FGLM-amide and SSSR, respectively). Although the path from the laboratory to the clinic in these instances has been relatively short, the time required to establish the newly identified treatment modalities in the wider community has been long. In this review, I relate the trajectory of my translational research career.     

Effects of substance P and IGF-1 in corneal epithelial barrier function and wound healing in a rat model of neurotrophic keratopathy

PURPOSE: To establish a rat model of neurotrophic keratopathy and to examine the effects of the combination of substance P (SP) and insulin-like growth factor (IGF)-1 on corneal epithelial barrier function and wound healing in this model. METHODS: Corneal denervation was achieved by thermocoagulation of the ophthalmic branch of the trigeminal nerve. A modified Schirmer test was performed without topical anesthesia. Corneal epithelial barrier function was assessed by measurement of fluorescein permeability with an anterior fluorophotometer. Epithelial wound healing was evaluated by measurement of the area of the defect at various times after removal of the entire epithelium. Eye drops containing both 1 mM SP and IGF-1 (1 micro g/mL) were administered six times daily. RESULTS: The Schirmer test result in eyes subjected to trigeminal denervation was lower than that in control eyes. The fluorescein permeability of the corneal epithelium of denervated eyes was increased relative to that of control eyes. Furthermore, trigeminal denervation induced a delay in corneal epithelial wound healing. Application of eye drops containing SP and IGF-1 to denervated corneas restored the fluorescein permeability of the corneal epithelium to control levels and abolished the delay in epithelial wound healing. CONCLUSIONS: A rat model of neurotrophic keratopathy, characterized by reduced tear secretion, loss of corneal sensation, impaired epithelial barrier function, and delayed epithelial wound healing, was established by trigeminal denervation. Treatment with both SP and IGF-1 improved corneal epithelial barrier function and stimulated corneal epithelial wound healing in this model.     

Participation of p38 MAP kinase, but not p44/42 MAP kinase, in stimulation of corneal epithelial migration by substance P and IGF-1

PURPOSE: Substance P and insulin-like growth factor-1 (IGF-1) synergistically promote corneal epithelial migration both in vitro and in vivo. The mechanism of this action was investigated. METHODS: The effects of various inhibitors and activators of intracellular signaling pathways on corneal epithelial migration were examined by measuring the length of the migration path in rabbit corneal blocks in culture. RESULTS: Inhibitors of signaling by p38 or p44/42 isoforms of mitogen-activated protein (MAP) kinase or of phosphatidylinositol (PI) 3-kinase reduced the extent of spontaneous migration of the corneal epithelium, whereas modulators of signaling by cyclic AMP- or cyclic GMP-dependent protein kinases had no effect. The inhibitors of p38 MAP kinase and of PI 3-kinase also abolished the stimulatory effect of substance P and IGF-1 on epithelial migration, whereas inhibitors of signaling by p44/42 MAP kinase or modulators of cyclic nucleotide-dependent signaling did not. CONCLUSIONS: These results suggest that various signal transduction systems participate in spontaneous corneal epithelial migration as well as in the combined effect of substance P and IGF-1 on this process. In particular, although both p38 and p44/42 isoforms of MAP kinase appear to regulate spontaneous corneal epithelial migration, the stimulatory effect of substance P and IGF-1 appears to be mediated by p38 MAP kinase but not by p44/42 MAP kinase.     

Promotion of corneal epithelial wound healing in vitro and in vivo by annexin A5

PURPOSE: To investigate the effect of annexin A5, a calcium-dependent phospholipid-binding protein, on corneal epithelial wound healing. METHODS: The effect of annexin A5 on migration of rabbit corneal epithelial (RCE) cells in vitro was examined in scrape-wounded cell monolayers. The effect of annexin A5 on the release of urokinase-type plasminogen activator (uPA) from cultured RCE cells was determined by zymography, fluorogenic assay of PA activity, and enzyme-linked immunosorbent assay. The proliferation of RCE cells was assessed by measurement of [3H]thymidine incorporation. The effect of annexin A5 on corneal wound closure in rabbits was investigated after removal of the corneal epithelium, either by exposure to iodine vapor or surgically. Eye drops containing annexin A5 were instilled into one eye and vehicle into the other. The area of the epithelial defect was measured at various times after wounding, and the healing rate was calculated by linear regression analysis. RESULTS: Annexin A5 significantly promoted the migration of RCE cells in a wounded monolayer. However, annexin A5 had no effect on RCE cell proliferation. Annexin A5 also increased the release of uPA both from wounded RCE cell monolayers and from nonwounded semiconfluent RCE cells. In both models of corneal wound closure, the healing rate was significantly increased by instillation of eye drops containing annexin A5 compared with that apparent in the eyes that received vehicle. CONCLUSIONS: Annexin A5 promoted corneal epithelial wound healing both in vitro and in vivo. Upregulation of uPA release from corneal epithelial cells may contribute to this effect of annexin A5.     

Spontaneous chronic corneal epithelial defects (SCCED) in dogs: clinical features, innervation, and effect of topical SP, with or without IGF-1

PURPOSE: To delineate the clinical features and alterations in innervation and substance P (SP) content in spontaneous chronic corneal epithelial defects (SCCED) in dogs and to conduct a preliminary investigation evaluating the efficacy of topical SP, with or without insulin-like growth factor (IGF)-1, in the treatment of this disorder. METHODS: Complete ophthalmic examinations, including Cochet-Bonnet aesthesiometry, were performed in 45 canine patients that had spontaneous corneal epithelial defects of at least 3 weeks' duration and with no identifiable cause. Eighteen patients had superficial keratectomies performed, and the corneal nerves were labeled immunohistochemically with antibodies against protein gene product (PGP)-9.5, SP, vasoactive intestinal peptide (VIP), and tyrosine hydroxylase (TH). Relative fiber densities were assessed qualitatively and quantitatively. Corneal epithelial cell and tear SP contents were determined in affected and normal dogs by an enzyme immunoassay. A preliminary open-label treatment trial of topical SP, with and without IGF-1, was conducted in 21 dogs. RESULTS: The duration of the erosion before admittance into the study was a mean of 9.22 weeks (range, 3-52). The average patient was middle aged (mean, 9.25 +/- 1.85 years [SD]); no sex predisposition of the disease was identified. Boxers, golden retrievers, and keeshonds were overrepresented when compared with the normal hospital population. Corneal sensation was normal. Marked alterations in corneal innervation were identified in affected dogs with abnormal increased SP and calcitonin gene-related peptide (CGRP)-immunoreactive nerve plexuses identified surrounding the periphery of the epithelial defect. The SP content of epithelial cells surrounding the defect increased, whereas the tear SP content remained unchanged. Of the canine patients treated with SP, with or without IGF-1, 70% to 75% had complete healing of the defect. CONCLUSIONS: This idiopathic spontaneous corneal disease in dogs shares clinical features with chronic epithelial defects in humans. The presence of marked alterations in peptidergic innervation and positive response to topical therapy with SP suggest that SP plays a critical role in corneal wound-healing processes.     

Insulin, not leptin, promotes in vitro cell migration to heal monolayer wounds in human corneal epithelium

PURPOSE: To investigate the effects of insulin and leptin on in vitro wound healing of transformed human corneal epithelial cell monolayers and to identify cellular (migration versus proliferation) and intracellular signaling mechanisms. METHODS: Scratch wounds were created in monolayers of an immortalized human corneal epithelial cell (HCEC) line. The wounded monolayers were exposed to insulin and leptin. Wound areas were measured every hour after wounding for up to 8 hours. Phosphoinositide 3-kinase (PI3-kinase) and mitogen-activated protein (MAP)-kinase signaling was analyzed with Western blot. The actions of insulin were also examined after incubation with inhibitors to extracellular signal regulated kinase (ERK 1/2) and PI3-kinase. RESULTS: The presence of insulin, but not leptin facilitated closure of wounds created in corneal epithelial cell monolayers. Phosphorylation of ERK 1/2 and Akt was stimulated after exposure of the monolayers to insulin. Inhibitors of PI3-kinase and ERK 1/2 prevented or reduced insulin-induced corneal wound healing, respectively. CONCLUSIONS: Exposure of corneal epithelium to insulin facilitated closure of in vitro small wounds through enhanced cell migration instead of proliferation, which depended on ERK 1/2 and PI3-kinase signaling. These data suggest a mechanism by which insulin may influence corneal wound healing in vitro. In vivo, disruptions to the insulin signaling pathway observed in diseases such as diabetes might account for the delayed wound healing and corneal defects.     

Potentiation by cyclic AMP of the stimulatory effect of epidermal growth factor on corneal epithelial migration

PURPOSE: To provide insight into the mechanism by which epidermal growth factor (EGF) stimulates corneal epithelial migration, we investigated the possible interaction between EGF and cyclic AMP (cAMP) signaling pathways during epithelial migration with an organ culture system for the rabbit cornea. METHODS: Rabbit corneal blocks were cultured in the absence or presence of various agents for 24 hours and were then fixed, dehydrated, embedded in paraffin, sectioned, and stained with hematoxylin-eosin. The path length of epithelial migration was measured on light micrographs of the stained sections. RESULTS: Epidermal growth factor alone stimulated corneal epithelial migration in a dose-dependent manner. In contrast, neither of two cell-permeable cAMP analogs, dibutyryl cAMP and 8-bromo cAMP, affected epithelial migration at concentrations up to 1 mM. In the presence of EGF (10 ng/mL), however, each of the two cAMP derivatives increased the extent of epithelial migration in a concentration-dependent manner. Neither the adenylate cyclase activator forskolin nor the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine alone affected epithelial migration. However, each of these two agents potentiated the stimulatory effect of EGF on this process. The stimulatory effects of fibronectin, hyaluronan, and interleukin-6 on corneal epithelial migration were not modified by either dibutyryl cAMP or 3-isobutyl-1-methylxanthine. CONCLUSION: These results demonstrate that cAMP potentiates the stimulation of corneal epithelial migration by EGF in vitro, suggesting that endogenous cAMP might function as a modulator of epithelial wound healing promoted by this growth factor in vivo.     

Delay of corneal epithelial wound healing and induction of keratocyte apoptosis by platelet-activating factor

PURPOSE: To examine the role of the lipid mediator platelet-activating factor (PAF) in epithelial wound healing. METHODS: A 7-mm central de-epithelializing wound was produced in rabbit corneas, and the tissue was incubated with 125 nM carbamyl PAF (cPAF), an analogue of PAF. Rabbit corneal epithelial and stromal cells were also cultured in the presence of cPAF. Cell adhesion, proliferation, and migration assays were conducted. Apoptosis was assayed by TUNEL staining on preparations of corneal tissue sections and in cells in culture. RESULTS: Twenty-four hours after injury, 50% of the wounded area was covered by new epithelium, whereas only 30% was covered in the presence of cPAF. At 48 hours, the epithelium completely closed the wound, but only 45% of the original wound was covered in corneas treated with cPAF. Similar inhibition of epithelial wound closure was found with human corneas incubated with PAF in organ culture. Moreover, addition of several growth factors involved in corneal wound healing, such as epidermal growth factor, hepatocyte growth factor, and keratinocyte growth factor, could not overcome the inhibitory action of PAF in wound closure. Three PAF antagonists, BN50727, BN50730, and BN50739, abolished the effect of PAF. A significant increase in TUNEL-positive staining occurred in corneal stromal cells (keratocytes), which was inhibited by preincubating the corneas with PAF antagonists. However, no TUNEL-positive staining was found in epithelial cells. TUNEL-staining results in cultured stromal cells (keratocytes) and epithelial cells in first-passage cell culture were similar to those in organ-cultured corneas. In addition, PAF caused 35% to 56% inhibition of adhesion of epithelial cells to proteins of the extracellular matrix: collagen I and IV, fibronectin, and laminin. There were no significant changes in proliferation or migration of epithelial cells induced by the lipid mediator. CONCLUSIONS: The results suggest PAF plays an important role in preventing corneal wound healing by affecting adhesion of epithelial cells and increasing apoptosis in stromal cells. PAF antagonists could be of therapeutic importance during prolonged ocular inflammation, helping to avoid loss of corneal transparency and visual acuity.     

Characterization of wound reepithelialization using a new human tissue-engineered corneal wound healing model

PURPOSE: The reepithelialization of the corneal surface is an important process for restoring the imaging properties of this tissue. The purpose of the present study was to characterize and validate a new human in vitro three-dimensional corneal wound healing model by studying the expression of basement membrane components and integrin subunits that play important roles during epithelial cell migration and to verify whether the presence of exogenous factors could accelerate the reepithelialization. METHODS: Tissue-engineered human cornea was wounded with a 6-mm biopsy punch, and the reepithelialization from the surrounding margins was studied. Biopsy samples of the reepithelialized surface were harvested 3 days after wounding and were processed for histologic, electron microscopic, and immunofluorescence analyses. The effects of fibrin and epithelial growth factor (EGF) on wound reepithelialization were also studied. RESULTS: Results demonstrated that this in vitro model allowed the migration of human corneal epithelial cells on a natural extracellular matrix. During reepithelialization, epithelial cell migration followed a consistent wavelike pattern similar to that reported for human corneal wound healing in vivo. This model showed a histologic appearance similar to that of native tissue as well as expression and modulation of basement membrane components and the integrin subunits known to be main actors during the wound healing process. It also allowed quantification of the reepithelialization rate, which was significantly accelerated in the presence of fibrin or EGF. The results indicated that alpha v beta6 integrin expression was increased in the migrating epithelial cells compared with the surrounding corneal tissue. CONCLUSIONS: The similarity observed with the in vivo wound healing process supports the use of this tissue-engineered model for investigating the basic mechanisms involved in corneal reepithelialization. Moreover, this model may also be used as a tool to screen agents that affect reepithelialization or to evaluate the effect of growth factors before animal testing.