epidermal-growth-factor and Corneal-Injuries

Agonist and antagonist drugs acting on epidermal growth factor receptor (EGFR) signaling are emerging as a new possibility for pharmaceutical study and clinical manipulation of some skin and corneal disorders. EGFR activation appears to be effective in reducing the time of reepithelialization after corneal wound healing, with potential uses in penetrating keratoplasty, refractive surgery, alkali burns, diabetic keratopathy, keratopathy following chemotherapy, cornea transplantation, and dry eye. Most of the studies show therapeutic advantages of human recombinant epidermal growth factor (hrEGF) eye drops without showing adverse effects. In contrast, EGFR inhibition delays epithelial cell proliferation and stratification during corneal regeneration.The aim of this review is to summarize the most seminal discoveries and recent advances so as to clarify the role of the EGFR system in corneal physiology and pharmacology. Epidermal growth factor eye drops could be a first-choice treatment for promoting regeneration in numerous epithelial defects in the medium to long term.

Topics: Animals; Cell Proliferation; Cornea; Corneal Injuries; Epidermal Growth Factor; Epithelium, Corneal; ErbB Receptors; Humans; Recombinant Proteins; Regeneration; Signal Transduction; Wound Healing

A number of growth factors and their associated receptors, including epidermal growth factor, transforming growth factor-beta, keratinocyte growth factor, hepatocyte growth factor, fibroblast growth factor and platelet-derived growth factor have been detected in the anterior segment of the eye. On binding to cellular receptors, these factors activate signalling cascades, which regulate functions including mitosis, differentiation, motility and apoptosis. Production of growth factors by corneal cells and their presence in the tear fluid and aqueous humour is essential for maintenance and renewal of normal tissue in the anterior eye and the prevention of undesirable immune or angiogenic reactions. Growth factors also play a vital role in corneal wound healing, mediating the proliferation of epithelial and stromal tissue and affecting the remodelling of the extracellular matrix (ECM). These functions depend on a complex interplay between growth factors of different types, the ECM, and regulatory mechanisms of the affected cells. Imbalances may lead to deficient wound healing and various ocular pathologies, including edema, neovascularization and glaucoma. Growth factors may be targeted in therapeutic ophthalmic applications, through exogenous application or selective inhibition, and may be used to elicit specific cellular responses to ophthalmic materials. A thorough understanding of the mechanism and function of growth factors and their actions in the complex environment of the anterior eye is required for these purposes. Growth factors, their function and mechanisms of action as well as the interplay between different growth factors based on recent in vitro and in vivo studies are presented.

Topics: Animals; Aqueous Humor; Cornea; Corneal Injuries; Epidermal Growth Factor; Epithelial Cells; Extracellular Matrix; Fibroblast Growth Factor 7; Fibroblast Growth Factors; Growth Substances; Hepatocyte Growth Factor; Humans; Nerve Growth Factors; Platelet-Derived Growth Factor; Tissue Engineering; Transforming Growth Factor beta; Wound Healing

The mechanism of corneal wound healing has not been clarified yet. However, evidence has accumulated that various kinds of growth factor such as epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF) play a key role in corneal wound healing. For example, these growth factors are expressed in the corneal epithelial cells, keratocytes and endothelial cells, and their receptors are expressed in the corneal cells. Furthermore, these growth factors promote the proliferation of corneal cells and induce the migration of corneal cells. In addition to the growth factors, inflammatory cytokines such as interleukin (IL)-1, IL-6 and TNF-alpha are involved in corneal wound healing. These cytokines are expressed in the normal and inflammatory cornea after infections, alkaliburn, etc. where they control the growth of corneal cells and induce the migration of corneal cells. Thus, a number of growth factors and cytokines function in the regulation of corneal cell proliferation and in the maintenance of corneal transparency.

Topics: Animals; Cell Movement; Corneal Injuries; Cytokines; Epidermal Growth Factor; Growth Substances; Humans; In Vitro Techniques; Platelet-Derived Growth Factor; Transforming Growth Factor beta; Wound Healing

Protein growth factors regulate many of the processes in vitro that are essential for the process of normal ocular wound healing, including migration, mitosis and differentiation of cells. This has led to the hypothesis that peptide growth factors play key roles in regulating normal ocular wound healing in vivo. A corollary to this concept is that insufficient action of growth factors causes impaired healing, and prolonged action of growth factors produces excessive scarring. If both of these concepts are correct, then the addition of exogenous protein growth factors should enhance healing of chronic ocular wounds and reducing prolonged actions of growth factors should limit excessive scarring. Although much remains to be understood about the role of growth factors in ocular development and wound healing, results of a substantial number of laboratory and clinical experiments indicate that these hypotheses are generally correct. This article reviews the results of pre-clinical experiments and clinical trials investigating the roles of protein growth factors in ocular development and wound healing.

Topics: Animals; Corneal Injuries; Endothelium, Corneal; Epidermal Growth Factor; Eye Injuries; Growth Substances; Humans; Mice; Rabbits; Transforming Growth Factor alpha; Wound Healing

Wound healing is a complex, long-lasting regulatory sequence that involves expression of a number of genes, which are active during the individual's development. Some of the phenomena differ from normal tissue turnover and growth only quantitatively. This article reviews the current data on corneal wound healing, with particular reference to mesenchymal matrix proteins and their integrin receptors, to growth factors and to proteolytic enzymes. Some inflammatory mediators are also discussed. The theoretical basis for therapeutic interventions is also discussed briefly, in the light of present knowledge.

Topics: Cell Adhesion; Cornea; Corneal Injuries; Epidermal Growth Factor; Epithelium; Extracellular Matrix Proteins; Eye Injuries; Eye Proteins; Humans; Integrins; Serine Endopeptidases; Wound Healing

Topics: Animals; Cornea; Corneal Injuries; Epidermal Growth Factor; Fibroblast Growth Factors; Growth Substances; Humans; Organ Culture Techniques; Submandibular Gland; Wound Healing

The tolerability and efficacy of epidermal growth factor (EGF) in the topical treatment of traumatic corneal ulcers have been evaluated in a double-blind, placebo-controlled, randomized study in two groups of patients. The time required for complete reepithelialization of the cornea was recorded, and the data obtained were analyzed statistically. In the EGF group the reepithelialization was significantly faster than in the control group. Tolerability of EGF was always excellent. These results indicate that EGF is safe and effective in reducing the healing time in the management of superficial corneal lesions.

Topics: Administration, Topical; Adolescent; Adult; Aged; Cornea; Corneal Injuries; Corneal Ulcer; Double-Blind Method; Epidermal Growth Factor; Epithelium; Female; Humans; Male; Middle Aged; Ophthalmic Solutions; Wound Healing

A randomized, double-blind, placebo-controlled, multicenter clinical trial was conducted in five centers to assess safety, ocular tolerance, and efficacy of an ophthalmic solution of epidermal growth factor (EGF) for the treatment of traumatic corneal epithelial defects. One hundred four patients completed the study: 47 received EGF and 57 placebo (the drug vehicle). Mean epithelial healing time was significantly enhanced in the EGF-treated group (44.17 h) compared with the placebo-treated treated group (61.05 h) (p < 0.01). The number of epithelial defects completely healed at 24, 48, and 72 h after the onset of treatment was significantly greater in the EGF-treated group. Local tolerance was adequate in both groups. These results indicated that topical EGF is well tolerated and may be a significant addition to the ophthalmologist's armamentarium for treating corneal epithelial defects.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Cornea; Corneal Injuries; Double-Blind Method; Epidermal Growth Factor; Epithelium; Eye Injuries; Female; Humans; Male; Middle Aged; Ophthalmic Solutions; Placebos; Wound Healing

To investigate the role of neutrophils in corneal nerve regeneration.. A mouse model simulating corneal nerve injury was established and samples from corneal scraping with and without neutrophil closure were collected. These samples were used for corneal nerve staining, ribonucleic acid sequencing, and bioinformatics. Differential expression analysis was used to perform enrichment analysis to identify any significant differences between these two groups. The differential genes were then intersected with neutrophil-associated genes and a protein-protein interaction network was constructed using the intersected genes. The immune infiltration between the two groups was examined along with the immune cell variation between the high and low gene expression groups.. Neutrophil removal delays corneal epithelial and nerve regeneration. A total of 546 differential genes and 980 neutrophil-associated genes, with 27 genes common to both sets were obtained. Molecular Complex Detection analysis yielded five key genes, namely integrin subunit beta 2 (ITGB2), matrix metallopeptidase 9 (MMP9), epidermal growth factor (EGF), serpin family E member 1 (SERPINE1), and plasminogen activator urokinase receptor (PLAUR). Among these genes, ITGB2, SERPINE1, and PLAUR exhibited increased expression in the neutrophil-confined group, while MMP9 and EGF showed decreased expression, with MMP9 and EGF displaying a more significant difference. Immune infiltration was also observed between the two groups, revealing significant differences in the infiltration of M0 macrophages, activated mast cells, and neutrophils. Moreover, the neutrophil levels were lower in the groups with low MMP9 and EGF expressions and higher in the high-expression group.. Neutrophil confinement might significantly affect the MMP9 and EGF expression levels. Strategies to inhibit MMP9 could potentially yield therapeutic benefits.

Topics: Animals; Cornea; Corneal Injuries; Epidermal Growth Factor; Matrix Metalloproteinase 9; Mice; Nerve Regeneration; Neutrophils

Oxidative stress is a major pathogenesis of some ocular surface diseases. Our previous study demonstrated that epidermal growth factor (EGF)-activated reactive oxygen species (ROS) could protect against human corneal epithelial cell (HCE) injury. In the present study, we aimed to explore the role and mechanisms of oxidative stress and mitochondrial autophagy in HCE cells subjected to scratch injury. CCK-8 assays, EdU assays, Western blot analysis, wound-healing assays, and flow cytometry were conducted to determine cell viability, proliferation, protein expression, cell apoptosis, and intracellular ROS levels, respectively. The results showed that EGF could promote damage repair and inhibit cell apoptosis in scratch injured HCE cells by upregulating ROS (**p < .01, ***p < .001). EGF also induced mitochondrial autophagy and alleviated mitochondrial damage. Interestingly, the combination of the mitochondrial autophagy inhibitor and mitochondrial division inhibitor 1 (MDIVI-1) with EGF could reduce cell proliferation, viability, and the ROS level (*p < .05, **p < .01, ***p < .001). Treatment using the ROS inhibitor N-acetyl- l-cysteine abrogated the increase in mitochondrial membrane potential after EGF treatment. (*p < .05). Taken together, these findings indicated that EGF plays an important role in HCE damage repair and could activate ROS to protect against HCE injury by inducing mitochondrial autophagy via activation of TRPM2.

Topics: Apoptosis; Autophagy; Cell Line; Cell Proliferation; Cell Survival; Cornea; Corneal Injuries; Epidermal Growth Factor; Epithelial Cells; Humans; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Dynamics; Oxidative Stress; Reactive Oxygen Species; TRPM Cation Channels

We aimed to determine and compare the in vitro effects of autologous bone marrow-derived mesenchymal stem cells (BM-MSCs) and mesenchymal stem cell supernatant (MSC-Sp) on the wound healing capacity of equine corneal fibroblasts using a scratch assay.. There was a significant percentage decrease in the scratch area remaining in the BM-MSC and MSC-Sp groups compared to the control group. There was also a significant percentage decrease in the scratch area remaining in the BM-MSC group compared to the MSC-Sp group at 36 h post-scratch and all time points thereafter. The concentration of transforming growth factor (TGF)-β1 in the media was significantly higher in the BM-MSC group compared to the control group.. The significant decrease in scratch area in equine corneal fibroblast cultures treated with autologous BM-MSCs compared to MSC-Sp or control treatments suggests that BM-MSCs may substantially improve corneal wound healing in horses. MSC-Sp may also improve corneal wound healing given the significant decrease in scratch area compared to control treatments, and would be an immediately available and cost-effective treatment option.

Topics: Animals; Biomarkers; Bone Marrow Cells; Corneal Injuries; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Female; Flow Cytometry; Horses; Male; Mesenchymal Stem Cells; Transforming Growth Factor beta1; Wound Healing

Limbal stem cell (LSC) on the basal layer of cornea plays an important role in the epithelial repair after corneal injury as it can proliferate, differentiate and migrate into injury sites under the direction of cytokines. This study explored the signaling pathway and cellular mechanism between corneal epithelial cells LSC, on a mouse model with mechanic corneal injury. Ipsilateral corneal mechanic injury model was prepared on mice using the contralateral eye as the control. Tissues from both central and peripheral regions of cornea were collected, cultured and quantified for expression of various cytokines including epidermal growth factor (EGF), fibroblast growth factor-β (FGF-β), heparin-like growth factor (HGF), keratinocyte growth factor (KGF), transforming growth factor-β1 (TGF-β1), IGF-1 and IGF-2. The effects of these factors on the differentiation of LSC and fibroblasts were also studied. Most of those cytokines had elevated gene expressions after the corneal injury. Among those IGF-2 had significantly increased expression, along with the high expression of IGF-2 receptor in corneal peripheral cells. IGF-2 also induced the differentiation of LSC into keratin-12-positive cells. Further studies showed the prominent expression of α-actin in injured tissues, suggesting the potential transformation of fibroblasts into myofibroblasts. Both IGF-2 and its receptor had elevated expressions after corneal injury. They may facilitate the transformation of LSC into epithelial cells, in addition to the role in transformation from fibroblasts to myofibroblasts.

Topics: Animals; Cornea; Corneal Injuries; Epidermal Growth Factor; Fibroblast Growth Factor 7; Fibroblast Growth Factors; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Male; Mice; Mice, Inbred BALB C; Receptor, IGF Type 2; Wound Healing

The purpose of this study was to evaluate changes in tear volume, epidermal growth factor (EGF), and histology of the rabbit lacrimal gland after intraglandular application of botulinum toxin.. Ten New Zealand rabbits were separated into 3 groups. Eight rabbits received botulinum toxin injection (2.5 U per 0.1 mL) into the right lacrimal gland and saline injection (0.1 mL) into the left lacrimal gland as a sham control. Two rabbits were untreated to serve as normal controls. Tear volume was measured using cotton thread every 2 weeks, and 4 rabbits were killed after 2 and 4 weeks. The lacrimal glands were surgically excised and sectioned or lysed for gene expression analysis. Epidermal growth factor expression and concentration were evaluated by real-time polymerase chain reaction and enzyme-linked immunosorbent assay; morphometric and histologic analyses were performed.. The rabbits that were killed 2 weeks after the injection showed decreased tear volume and increased EGF expression and concentration, but differences were not statistically significant. The rabbits that were killed 4 weeks after the injection exhibited similar results. When all 8 rabbits were considered, we detected a significant decrease in tear volume and increased EGF expression and concentration (P = 0.012, P = 0.011, and P = 0.012, respectively). The EGF level was not significantly correlated with the tear volume. There were no prominent histologic changes between the glands, and the lumen versus fibrosis ratio in the interlobular ducts showed no statistically significant difference.. The use of botulinum toxin in patients with epiphora is effective, safe, and repeatable because it reduces tear volume and increases the EGF level to prevent corneal damage while causing no histologic changes.

Topics: Acetylcholine Release Inhibitors; Animals; Botulinum Toxins; Corneal Injuries; Elastic Tissue; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Gene Expression Profiling; Lacrimal Apparatus; Lacrimal Apparatus Diseases; Male; Rabbits; Real-Time Polymerase Chain Reaction; Tears; Time Factors

EGF-induced activation of the epigenetic CCCTC binding factor (CTCF) plays an important role in corneal epithelial cell proliferation by suppressing the Pax6 gene. The present study focused on further understanding the role of CTCF in mediating EGF-induced migration of immortalized human corneal epithelial cells.. CTCF activities in human corneal epithelial cells immortalized by telomerase (HTCE cells) and SV-40 (HCE cells) transformation were suppressed and enhanced by CTCF mRNA knockdown and by overexpressing CTCF cDNA, respectively. EGF-induced cell migration was evaluated by linear scratch wound healing, a cell migration assay, and live cell motility GFP-tracking with a fluorescence microscope. Immunochemical analysis was performed for detecting focal adhesion changes in EGF-induced and CTCF activity-altered cells.. EGF-induced wound closure and cell migration rates of human corneal epithelial cells were significantly suppressed and enhanced by CTCF mRNA knockdown and by overexpression of CTCF, respectively. CTCF mRNA knockdown also markedly suppressed cell motility, determined by using a live-cell-tracking system in GFP-tag-expressed HTCE cells. Finally, alterations of EGF-stimulated focal adhesion were observed in CTCF knockdown HTCE cells by immunostaining of F-actin and vinculin in cytoskeleton reorganization.. CTCF, an epigenetic regulator and transcription factor, involves EGF-induced increases in cell motility and migration. CTCF plays an essential role in growth factor-regulated human corneal epithelial cell wound healing.

Topics: Blotting, Western; CCCTC-Binding Factor; Cell Movement; Cell Proliferation; Cornea; Corneal Injuries; Epidermal Growth Factor; Epithelium, Corneal; Follow-Up Studies; Gene Expression Regulation; Humans; Immunohistochemistry; Microscopy, Fluorescence; Polymerase Chain Reaction; Repressor Proteins; RNA, Messenger; Telomerase; Wound Healing; Zinc Fingers

To evaluate the therapeutic effects of epidermal growth factor (EGF) combined with plasma cryoprecipitate (CRYO) on the corneal injury induced by paraquat (PQ).. According to the "Toxicological test methods of pesticides for registration" (GB 15670-1995), the conjunctival sacs of 18 health New Zealand rabbits were exposed to 100 µl 20% PQ, which were randomly divided into EGF, CRYO and EGF plus CRYO groups. The routine treatments (normal saline washing and antibiotic eyedrops) were administrated to the injured eyes of 3 groups, at the same time the left eyes of 3 groups were treated with EGF, CRYO and EGF plus CRYO, respectively. The injury of conjunctival, iris and corneal, fluorescent stranded and pathology changes of corneal were observed. The injury score was calculated and the recovery time of corneal injury was recorded.. The recovery time of corneal injury in EGF and EGF plus CRYO groups were 19.50 ± 3.08 and 18.67 ± 2.73 days, respectively which were significantly lower than those (27.33 ± 2.58 and 26.83 ± 3.13 days) in corresponding routine treatment controls (P < 0.05).. EGF and EGF plus CRYO could be used to treat the corneal injury induced by paraquat.

Topics: Animals; Blood Transfusion, Autologous; Cornea; Corneal Injuries; Epidermal Growth Factor; Eye Injuries; Factor VIII; Fibrinogen; Ophthalmic Solutions; Paraquat; Plasma; Rabbits; Treatment Outcome; Wound Healing

Heparin-binding EGF-like growth factor (HB-EGF) contains, in contrast to EGF, a domain that binds to negatively charged glycans on cell surfaces and in extracellular matrix. We speculated that a short exposure to HB-EGF induces prolonged biological effects such as healing of wounds after immobilization in tissues.. Epithelial cell sheets in tissue and corneas in organ culture were treated briefly with HB-EGF or EGF and binding of the growth factors, time course of activation of the EGF receptor, and healing of wounds were compared.. Treating human corneal epithelial cells for 2 min with HB-EGF resulted in 8h of detectable activation of the EGF receptor, but activation was much shorter after EGF treatment. A brief treatment with HB-EGF, but not with EGF, induced significant acceleration of healing in wounds in epithelial sheets in tissue and organ culture. Bound HB-EGF was detectable up to 16 h after brief treatments. Neutralizing antibodies added after HB-EGF treatment blocked acceleration of healing, demonstrating the role of bound HB-EGF in accelerating healing.. A brief exposure to HB-EGF, but not to EGF, is sufficient to induce prolonged activation of the EGF receptor and to enhance healing.. Bound HB-EGF can serve as a pool that induces prolonged activation of the EGF receptor. EGF has been used experimentally to treat poorly healing wounds, but the frequent applications that are necessary have hampered its use clinically. The findings imply that HB-EGF may be a useful long-acting alternative to EGF.

Topics: Animals; Corneal Injuries; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Organ Culture Techniques; Rabbits; Wound Healing

EGF is an essential growth factor needed for epithelial cell proliferation and wound healing of the cornea, but the molecular mechanism is not understood. Although studies have shown that EGF in some non-phagocytic cells induces ROS generation, little is known about the role of ROS in corneal epithelial cells. Therefore, we examined the potential physiological role of ROS in corneal cell proliferation, adhesion and wound healing using rabbit or human corneal epithelial cells, and pig whole cornea organ culture as models. EGF (5 ng/ml)-induced ROS in serum-starved RCE or HCE cells were captured as DCFH fluorescence and detected by confocal microscopy. The elevation of ROS was eradicated when the cells were pretreated with an antioxidant N-acetylcysteine (NAC) or mannitol, or with inhibitor to NADPH oxidase (DPI), or to lipoxygenase (NDGA). EGF-induced ROS generation correlated with cell growth and activation of Akt and MAPK signaling pathways, while NAC eliminated all these effects. EGF-stimulated cell adhesion or migration in cell culture was greatly suppressed in the presence of NAC while EGF-facilitated epithelial cell wound healing in corneal organ culture was also blocked by NAC. This is the first demonstration of a novel ROS physiological function in corneal wound healing.

Topics: Animals; Antioxidants; Cell Adhesion; Cell Movement; Cell Proliferation; Cells, Cultured; Cornea; Corneal Injuries; Epidermal Growth Factor; Epithelial Cells; Epithelium, Corneal; Humans; Microscopy, Confocal; Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-akt; Rabbits; Reactive Oxygen Species; Recombinant Proteins; Sus scrofa; Wound Healing

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.. 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 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.. 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.

Topics: Basement Membrane; Cells, Cultured; Corneal Injuries; Epidermal Growth Factor; Epithelium, Corneal; Fibrin; Fibroblasts; Fluorescent Antibody Technique, Indirect; Humans; Integrins; Models, Biological; Tissue Engineering; Wound Healing

The early events that occur rapidly after injury trigger signal cascades that are essential for proper wound closure of corneal epithelial cells. We hypothesize that injury releases ATP, which stimulates purinergic receptors and elicits the phosphorylation of epidermal growth factor receptor (EGFR) tyrosine residues and subsequent cell migration by a MMP and HB-EGF dependent pathway. We demonstrated that the inhibition of purinergic receptors with the antagonist, Reactive Blue 2, abrogated the phosphorylation of EGFR and ERK. Pre-incubation of cells with the EGFR kinase inhibitor, AG1478, and subsequent stimulation by injury or ATP resulted in a decrease in phosphorylation of EGFR and migration. Furthermore, downregulation of EGFR by siRNA, inhibited the EGF-induced intracellular Ca(2+) wave. However, the response to injury and ATP was retained indicating the presence of two signaling pathways. Inhibition with either CRM197 or TIMP-3 decreased injury and nucleotide-induced phosphorylation of both EGFR and ERK. Incubation in the presence of a functional blocking antibody to HB-EGF also resulted in a decrease in the phosphorylation of EGFR. In addition, cell migration was inhibited by CRM197 and rescued when cells were incubated with HB-EGF. We showed that injury-induced phosphorylation of specific tyrosine residues and found that a similar pattern of phosphorylation was induced by trinucleotides. These studies indicate that injury-induced purinergic receptor activation leads to phosphorylation of EGFR, ERK and migration.

Topics: Adenosine Triphosphate; Cell Line; Cell Movement; Corneal Injuries; Enzyme Inhibitors; Epidermal Growth Factor; Epithelial Cells; Epithelium, Corneal; ErbB Receptors; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Mitogen-Activated Protein Kinases; Nucleotides; Phosphorylation; Receptors, Purinergic; RNA, Small Interfering; Signal Transduction; Triazines; Wound Healing

Previous reports indicated that pregnancy and corneal injury (CI) trigger alterations of lacrimal gland (LG) growth factor expression and redistributions of lymphocytes from periductal foci to acini. The purpose of this study was to test our hypothesis that pregnancy would exacerbate the changes induced by CI.. Corneas were injured with scalpel blades, and, 2 weeks later, LGs were collected for immunocytochemistry and Western blot analysis. Lacrimal fluid was collected under basal- and pilocarpine-stimulated conditions for protein determination and Western blot analyses.. There were significant increases of immunoreactivity for prolactin, TGF-beta1, and EGF in duct cells during pregnancy and after CI, most prominent in pregnant animals with CI. Pregnancy decreased baseline lacrimal fluid secretion, whereas CI did not have a noticeable effect; pregnancy and CI combined resulted in increased fluid production. Pregnancy and CI each increased pilocarpine-induced lacrimal fluid production, whereas protein concentrations were decreased. Prolactin, TGF-beta1, and EGF were detected in LG by Western blot analysis but were minimally detectable in lacrimal fluid. RTLA+ and CD18+ cells were redistributed from periductal to interacinar sites during pregnancy and after CI, most prominent in pregnant animals with CI.. Like pregnancy, CI is associated with redistribution of immune cells from periductal to interacinar sites and enhanced immunoreactivity of prolactin, TGF-beta1, and EGF in ductal cells. Although baseline lacrimal fluid secretion varied, the glands of all three experimental groups produced significant amounts of fluid in response to pilocarpine, but protein concentrations were decreased.

Topics: Animals; Blotting, Western; Cornea; Corneal Injuries; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; Eye Injuries, Penetrating; Female; Immunohistochemistry; Lacrimal Apparatus; Muscarinic Agonists; Pilocarpine; Pregnancy; Pregnancy, Animal; Prolactin; Rabbits; T-Lymphocytes; Transforming Growth Factor beta; Transforming Growth Factor beta1

Our goal was to evaluate the role of epidermal growth factor and injury on the expression of integrin subunits alpha6(alpha6) and beta4(beta4). An in vitro wound model was used to evaluate corneal wound repair and cellular migration. Primary rabbit corneal epithelial cell cultures were serum-starved and injured in the presence or absence of EGF or tyrphostin AG1478, an inhibitor of EGF receptor kinase activity. Repair was monitored morphologically and expression was analyzed using in situ hybridization and immunohistochemistry accompanied by confocal microscopy. The addition of EGF to cell cultures induced a dose-dependent increase in beta4 mRNA expression but the constitutive expression of alpha6 was several fold greater. In the wounded cultures there was a rapid change in expression at the edge of the wound that was enhanced with EGF. In our model there was an increase in beta4 and alpha6 protein in migrating cells. Changes in integrin expression were accompanied by a transient increase in activation of the EGF receptor. The addition of tyrphostin inhibited migration of cells and wound repair, the activation of the EGF receptor and phosphorylation of beta4 in the cytoplasm. These data indicate that the activation of the EGF receptor plays a critical role in the regulation of integrin receptors and the mediation of cellular migration.

Topics: Animals; Antigens, Surface; Base Sequence; Cells, Cultured; Cornea; Corneal Injuries; DNA Primers; Epidermal Growth Factor; Epithelial Cells; Gene Expression Regulation; Immunohistochemistry; In Situ Hybridization; Integrin alpha6beta4; Integrins; Rabbits; RNA, Messenger

To investigate the effect of artificial tear (AT) solution and epidermal growth factor (EGF) treatment on the cornea and aqueous humour ascorbic acid (AA) levels of full-thickness corneal wounded eyes.. The effect of EGF on the AA levels of aqueous humour and corneal wound tissue was determined in full-thickness corneal wounded rabbit eyes on the seventh post-operative day. There were three groups: untreated controls, AT-treated controls and an EGF+AT-treated experimental group (n = 6 in each group). Corneal wounded eyes were topically treated with 5 microl AT or 5 microl EGF in AT (1 mg/l EGF in AT prepaaration which contained 3.0% carbopol 940) twice daily for 6 days after operation. The wound strengths were also measured on the seventh post-operative day as a measure of wound healing. Statistical analysis was carried out using the Mann-Whitney U-test by Statview program.. The wound strengths of corneas, and AA levels of wound tissues and aqueous humour, increased significantly following AT and EGF treatment (p < 0.05).. In the corneal wounded eye, aqueous humour serves as a source of vitamin C and there may be a relation between EGF treatment in AT and AA levels of corneal wounded eye tissues.

Topics: Animals; Aqueous Humor; Ascorbic Acid; Cornea; Corneal Injuries; Epidermal Growth Factor; Female; Male; Ophthalmic Solutions; Rabbits; Tensile Strength; Wound Healing

Topics: Animals; Corneal Injuries; Epidermal Growth Factor; Epithelium, Corneal; Eye Injuries; Female; Rabbits; Sucrose; Wound Healing

To investigate the effects of recombinant human epidermal growth factor (rhEGF) eye drops on corneal wound healing.. Twenty-four white rabbits were randomly divided into 4 groups, 6 rabbits 12 eyes each. Anterior keratectomy of 8 mm in diameter and 1/3 cornea in thickness was performed on each eye. Each of the following concentrations of rhEGF: 1, 10, 100 microg/ml eye drops or normal saline (control) was applied four times daily for a week respectively for one group. The wound area was determined by computer imaging analysis.. The mean epithelial healing rate of rhEGF 1, 10, 100 microg/ml groups was 9.31, 9.96, 9.31 mm(2)/day respectively, significantly greater than 8.11 mm(2)/day of the control group. The action of rhEGF of 10 microg/ml was somewhat better than that of 1 or 100 microg/ml, and no significant difference was noticed among the three rhEGF groups. Moderate inflammation and corneal neovascularization were induced in the rhEGF 100 microg/ml treated group.. rhEGF 1 - 10 microg/ml can accelerate corneal wound healing in the rabbit with no adverse side-effects. It may be used to treat serious corneal trauma and ulcer clinically.

Topics: Animals; Cornea; Corneal Injuries; Epidermal Growth Factor; Female; Inflammation; Male; Rabbits; Recombinant Proteins; Wound Healing

To determine whether there is an association between epidermal growth factor (EGF)-induced activation of phosphatidylinositol 3-kinase (PI 3-kinase) and stimulation of wound closure in rabbit corneal epithelial cells.. Immortalized rabbit corneal epithelial cells were cultured in 24-well plates until they became confluent. Circular wounds were created in confluent cultures by cell denudation and then incubated in the absence and presence of EGF for varying intervals. Wound closure was monitored by staining the cells with Giemsa and quantifying the wound area with SigmaS can computer program. Cell proliferation during wound repair was estimated by measuring the incorporation of [3H]thymidine into nuclear DNA. Changes in PI 3-kinase activity were assessed by measuring the production of phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] in 32P-labeled cells as well as by immunoprecipitating and assaying PI 3-kinase activity with phosphatidylinositol 4,5-bisphosphate and [gamma-32P]ATP as substrates. The enzyme product, PIP3, was analyzed by a combination of thin-layer and high-pressure liquid chromatography.. Addition of 10 ng/ml EGF to the wounded corneal epithelial cells stimulated wound closure in a time-dependent manner, and the wound closed completely within 48 hours. The effect of EGF was dose dependent, and maximal wound closure occurred at 10 ng/ml EGF. As the epithelial cells were undergoing EGF-stimulated wound closure, there was a time-dependent increase in PI 3-kinase activity. The enzyme activity increased maximally at 24 hours and then decreased gradually as the incubation was continued to 48 hours. When the cells were treated with wortmannin, a PI 3-kinase inhibitor, the EGF-stimulated PIP3 formation as well as the wound closure were inhibited significantly. Treatment of the cells with genistein or tyrphostin B42 also decreased both EGF-stimulated PIP3 formation and wound closure in a dose-dependent manner. Concomitant with stimulation of wound repair, the growth factor increased [3H]thymidine incorporation into nuclear DNA, and this effect was inhibited by pretreatment of the cell with wortmannin.. The data suggest a close correlation between EGF-stimulated wound closure and activation of PI 3-kinase in corneal epithelial cells. It can be concluded that PI 3-kinase might be an important component in signal transduction cascade initiated by EGF-receptor interaction, which leads to mitosis and cell proliferation during wound closure in corneal epithelial cells.

Topics: Androstadienes; Animals; Cell Line, Transformed; Cornea; Corneal Injuries; DNA; Enzyme Inhibitors; Epidermal Growth Factor; Epithelium; Phosphatidylinositol 3-Kinases; Phosphatidylinositol Phosphates; Phosphotransferases (Alcohol Group Acceptor); Protein-Tyrosine Kinases; Rabbits; Thymidine; Wortmannin; Wound Healing

The effects of growth factors on re-epithelialization of wounded human and bovine corneas were studied in a simple organ culture system. Excisional trephine and epithelial scrape wounds were created on bovine and human corneo-scleral rings in which the endothelial corneal concavity was then filled with an agar-collagen mixture. Organ culture was undertaken at 37 degrees C in a humidified 5% CO2 incubator with serum-free Medium 199 maintained at the level of the conjunctival epithelium. Rates of reepithelialization in response to addition of exogenous epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and transforming growth factor type beta 1 (TGF-beta 1) were assessed by image analysis. Corneal cultures could be maintained for up to 3 weeks without significant stromal oedema or keratocyte deterioration and with little loss of epithelial architecture. Following wounding the cornea reepithelialized in a similar fashion to that observed in vivo i.e. a lag phase followed by migration/proliferation and the reformation of an intact multilayered epithelium. EGF accelerated, basic FGF had no effect on, and TGF-beta 1 inhibited the rate of corneal re-epithelialization. Our organ culture model maintains corneal integrity and provides a practical system in which to study factors that modulate corneal reepithelialization following wounding.

Topics: Animals; Cattle; Cell Division; Cornea; Corneal Injuries; Dose-Response Relationship, Drug; Epidermal Growth Factor; Fibroblast Growth Factor 2; Growth Substances; Humans; Organ Culture Techniques; Time Factors; Transforming Growth Factor beta; Wound Healing

The effect of corneal epithelial wound creation on epidermal growth factor (EGF) concentration in tears was evaluated in order to better understand the effects of EGF on the wound healing process. The tears of New Zealand white rabbits were sampled by micropipette one day prior to wounding, immediately prior to the creation of a 7.5 mm diameter anterior keratectomy wound, immediately following wound creation, and at 1, 2, 3, 7, and 14 days following wounding. A volume of 50 microL was taken at each sampling time, and all tear samples were assayed for EGF by an enzyme linked immunosorbent assay (ELISA). The results demonstrated that the concentration of EGF in the tear layer rises dramatically immediately following wound creation. The basal measured EGF concentration was approximately 600 pg/mL; immediately following wound creation, this rose to approximately 1600 pg/mL. By 1 day following creation of the wound, the concentration of EGF in the tears had returned to the basal level. A second, marginally significant increase in the tear concentration was noted at 3 days post wounding. The EGF concentration in the tears were not significantly different at any other time. The measured dramatic rise in EGF concentration in the tears in response to the creation of a corneal epithelial wound provides further evidence of the importance of tear EGF in the wound healing process. The concentrations in all cases were on the order of ng/mL, suggesting that the intercellular concentrations in this range result in optimal cell stimulation.

Topics: Animals; Cornea; Corneal Injuries; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Epithelium; Rabbits; Tears; Wound Healing; Wounds, Nonpenetrating

To determine if there is a heterogeneous pattern of endothelin (ET) receptor subtype (i.e., ETA and ETB) gene expression in the bovine corneal epithelium (BCE). To determine if ET receptor subtype stimulation increases the effectiveness of epidermal growth factor (EGF) to accelerate wound closure in a primary culture of bovine corneal epithelial cells (BCEC).. In situ hybridization histochemistry was used to characterize ETA and ETB gene expression in the BCE. A wound closure assay evaluated wound healing rates in BCEC after 4 to 7 days in culture. [3H] thymidine incorporation and MTT assay measured proliferation.. ETA gene expression was appreciably higher in the basal cells than in the suprabasal cells, whereas the pattern for ETB was reversed. Epidermal growth factor (5 ng/ml) maximally increased wound closure by 145% above the control. With 5 ng/ml EGF, either 10(-9) M ET-1 or 10(-8) M sarafotoxin-6-c (s-6-c) increased wound closure by an additional 39% (P < 0.001) above that measured with 5 ng/ml EGF alone. BQ123 (10(-7) M) did not alter any of these effects of ET-1 or s-6-c. Epidermal growth factor stimulated wound closure through a selective increase in proliferation. Neither ET-1 nor s-6-c alone had any effect on proliferation or migration.. Both ETA and ETB genes are expressed in BCE. However, in BCEC only, ETB stimulation increases the effectiveness of EGF to stimulate wound closure. This response was caused by an increase in cell migration rather than proliferation because, after treatment with mitomycin C, neither ET-1 nor EGF stimulated wound closure.

Topics: Animals; Cattle; Cell Division; Cell Movement; Cells, Cultured; Cornea; Corneal Injuries; Endothelins; Epidermal Growth Factor; Epithelium; ErbB Receptors; In Situ Hybridization; Mitomycin; Receptors, Endothelin; Thymidine; Viper Venoms; Wound Healing

We propose a reaction-diffusion model of the mechanisms involved in the healing of corneal surface wounds. The model focuses on the stimulus for increased mitotic and migratory activity, specifically the role of epidermal growth factor. Analysis of the model equations elucidates the interaction and roles of the model parameters in determining the speed of healing and the shape of the traveling wave solutions which correspond to the migration of cells into the wound during the initial phase of healing. We determine an analytic approximation for the speed of traveling wave solutions of the model in terms of the parameters and verify the results numerically. By comparing the predicted speed with experimentally measured healing rates, we conclude that serum-derived factors can alone account for the overall features of the healing process, but that the supply of growth factors by the tear film in the absence of serum-derived factors is not sufficient to give the observed healing rate. Numerical solutions of the model equations also confirm the importance of both migration and mitosis for effective would healing. By modifying the model we obtain an analytic prediction for the healing rate of corneal surface wounds when epidermal growth factor is applied topically to the wound.

Topics: Animals; Cell Movement; Cornea; Corneal Injuries; Epidermal Growth Factor; Epithelium; Humans; Mitosis; Models, Biological; Models, Theoretical; Wound Healing

To determine whether epidermal growth factor (EGF) accelerates the healing of corneal tissue, we measured the tensile strength, ductility (total deformation at rupture) and toughness of 60 rabbit corneas with 7-mm perforating wounds after topical treatment with EGF or saline for 8, 13 or 23 days. The three mechanical properties were measured using corneal strips mounted on an Instron tensometer. A traction speed of 5 cm/min was chosen from speed-response curves for the three measures of interest. Since the values for eyes of a single rabbit were correlated, we used the data for one eye, selected at random, from each animal in the data analysis. EGF enhanced tensile strength (p = 0.003) and toughness (p = 0.03) of corneas without reducing ductility. The difference in tensile strength and toughness between EGF- and saline-treated corneas was more striking at 8 and 13 days than at 23 days. Histologic studies at 13 and 23 days supported these observations: at 13 days the healing process was more advanced in EGF-treated corneas, whereas at 23 days no histologic differences were noted. We conclude that EGF accelerates wound healing in rabbit corneas with perforating wounds without reducing ductility of the corneal tissue.

Topics: Administration, Topical; Animals; Cornea; Corneal Injuries; Elasticity; Epidermal Growth Factor; Eye Injuries, Penetrating; Female; Ophthalmic Solutions; Rabbits; Tensile Strength; Wound Healing

We investigated the effects of EGF, IL-1 and their combination on closure of wounds inflicted on rabbit corneal epithelial cell cultures and on migration of these cells in microchemotaxis chambers. In vitro corneal epithelial wound closure depended on the applied concentrations of EGF or IL-1. Twenty-four hours after wounding, the smallest wounds were obtained with 50 ng ml-1 of EGF and 1 ng ml-1 of IL-1, respectively. The effect on wound closure of combinations of EGF and IL-1 was additive even at concentrations that were optimal for each growth factor when applied alone. We found that EGF increases the chemotactic migration of rabbit corneal epithelial cells. Cell chemotaxis depended both on the concentration of EGF and on the number of cells applied in the assay. This response to EGF was seen at concentrations that were effective in the wound closure assay. The magnitude of the chemotactic migration response was much smaller with IL-1 than with EGF. Similarly to the observations on wound closure, the effect on cell chemotaxis of combinations of EGF and IL-1 was additive. The ability of EGF, and EGF/IL-1 combinations to modulate corneal epithelial cell chemotactic migration supports migration as a possible biological mechanism of the acceleration of corneal epithelium wound closure by these drugs.

Topics: Animals; Cell Count; Cells, Cultured; Chemotaxis; Cornea; Corneal Injuries; Dose-Response Relationship, Drug; Drug Synergism; Epidermal Growth Factor; Interleukin-1; Rabbits; Wound Healing

The authors investigated the mechanisms by which hyaluronic acid (HA) promotes corneal epithelial wound healing. The effect of HA on epithelial cell proliferation was examined in comparison with that of epidermal growth factor (EGF) and fibronectin (FN).. A penetrating linear incision (6-mm long) was made in the center of rabbit corneas. Immediately after wounding, the corneas were excised and cultured in TCM199 medium alone or medium containing HA (molecular weight, 860,000 Daltons; 100, 400, or 1000 micrograms/ml), EGF (25, 100, or 250 ng/ml), or FN (100 or 250 micrograms/ml) for 15 hr at 37 degrees C. The corneas were then labeled with tritiated thymidine (10 microCi/ml) and subjected to autoradiography.. In the corneas cultured with HA at concentrations of 400 and 1000 micrograms/ml, the number of epithelial cells incorporating tritiated thymidine was significantly higher than that in the control corneas. In the corneas cultured with EGF at all concentrations, it was also higher than that of the control. FN did not affect cell proliferation.. HA stimulates corneal epithelial cell proliferation. This stimulating effect of HA on epithelial cell proliferation might partly explain its effect in promoting corneal epithelial wound healing.

Topics: Animals; Autoradiography; Cell Division; Cornea; Corneal Injuries; DNA Replication; Epidermal Growth Factor; Epithelium; Fibronectins; Hyaluronic Acid; Organ Culture Techniques; Rabbits; Thymidine; Wound Healing

Healing of corneal alkali injuries remains a severe clinical challenge. The authors evaluated the effect of a new synthetic inhibitor of matrix metalloproteinases (GM6001 or N-[2(R)-2-(hydroxamido carbonylmethyl)-4-methylpentanoyl]-L-tryptophane methylamide) on preventing ulceration of rabbit corneas after alkali injury. Topical treatment of corneas with severe alkali injuries with 400 micrograms/ml or 40 micrograms/ml GM6001 alone prevented ulceration for 28 days, although 8 of 10 corneas treated with vehicle perforated. Corneas treated with 4 micrograms/ml GM6001 had midstromal depth ulcers. Corneas treated with 400 micrograms/ml of GM6001 contained very few inflammatory cells and had significantly reduced vessel ingrowth compared with vehicle-treated corneas. Epithelial regeneration after moderate alkali injuries also was investigated. Persistent epithelial defects developed 4 days after moderate alkali injury in rabbit corneas treated with vehicle and progressively increased to an average of 20% of the original 6 mm diameter wound by 27 days after moderate alkali injury. By contrast, epithelial regeneration was complete and persisted for 21 days for corneas treated with a formulation containing GM6001 (400 micrograms/ml), epidermal growth factor (10 micrograms/ml), fibronectin (500 micrograms/ml), and aprotinin (400 micrograms/ml). Sporadic punctate staining developed in 20% of the corneas treated with the combination of agents between days 21-28 after moderate alkali injury. These results demonstrate that topical application of GM6001 prevented corneal ulceration after severe alkali injury and that a combination containing GM6001, epidermal growth factor, fibronectin, and aprotinin promoted stable regeneration of corneal epithelium after moderate alkali injury.

Topics: Alkalies; Animals; Aprotinin; Burns, Chemical; Cornea; Corneal Injuries; Corneal Ulcer; Dipeptides; Dose-Response Relationship, Drug; Epidermal Growth Factor; Extracellular Matrix; Eye Burns; Fibronectins; Metalloendopeptidases; Rabbits; Regeneration

Topics: Animals; Calcium-Binding Proteins; Corneal Injuries; Epidermal Growth Factor; G1 Phase; Gene Expression Regulation; Rabbits; RNA, Messenger; S100 Proteins; Time Factors; Wound Healing

We identified the local treatment effect of epidermal growth factor on corneal wound healing of alloxan diabetic mice. The corneal wounds were induced by 0.5 M NaOH solution on the corneal surfaces of the diabetic and non-diabetic groups. The local epidermal growth factor solution (100 ng ml-1) was dropped in 5-microliters aliquots into the eye twice a day. The corneal wounds were measured daily for 15 days and examined histologically at the end of 15th day of experimental period. The results indicated that topical epidermal growth factor treatment of diabetic and non-diabetic mice greatly improved the incidence of healing of wounded corneas.

Topics: Administration, Topical; Animals; Cornea; Corneal Injuries; Diabetes Mellitus, Experimental; Epidermal Growth Factor; Female; Male; Mice; Time Factors; Wound Healing

Human recombinant epidermal growth factor (hEGF) was evaluated in various corneal wound healing models in the rabbit. Human EGF accelerated epithelial wound healing in corneal reepithelialization, anterior-keratectomy, and alkali-burn models at concentrations of 10-500 micrograms/ml given four times daily (qid). In the corneal reepithelialization model, 100 micrograms/ml of hEGF qid produced a 45% increase in the wound-healing rate compared with control (0.13 versus 0.09 mm/hr) with a similar response at 500 micrograms/ml qid. In the anterior-keratectomy model, 500 micrograms/ml of hEGF qid accelerated healing by 40% (0.07 versus 0.05 mm/hr), although the 100 micrograms/ml dose was not active in this model, and 1 microgram/ml of hEGF actually slowed the healing rate. In the alkali-burn model, 10 and 100 micrograms/ml of hEGF qid for 32 days appeared to produce faster initial healing of the wound compared with control, although the wound recurred in both hEGF and control groups. These results suggest that hEGF may be helpful in some epithelial disorders in humans, although considerations of dose response and optimal dosing regimens must be addressed.

Topics: Animals; Burns, Chemical; Cornea; Corneal Injuries; Dose-Response Relationship, Drug; Drug Evaluation; Epidermal Growth Factor; Epithelium; Eye Burns; Humans; Rabbits; Recombinant Proteins; Sodium Hydroxide; Wound Healing

Topics: Animals; Corneal Diseases; Corneal Injuries; Epidermal Growth Factor; Eye Injuries; Humans; Keratoconjunctivitis Sicca; Lacrimal Apparatus; Mice; Ocular Physiological Phenomena; Rabbits; Tears; Wound Healing

The proliferation and collagen synthesis of keratocytes during corneal wound healing after alkali-burn were investigated using 3H-thymidine or 3H-proline autoradiography. The effect of epidermal growth factor (EGF) on keratocytes was also examined. On day 1 after burn, the wounded stroma lacked keratocytes, and keratocytes at the periphery of the wound started to proliferate from the endothelial side. On day 7 and day 14, the population of keratocytes returned to normal. Collagen synthesis activity of the keratocytes was observed from day 7 through day 56, with the highest activity on around day 21. After day 14, keratocytes at the site of epithelial reopening or ulceration started to proliferate again. In the early stage of wound healing more keratocytes incorporated H-thymidine in eyes treated with EGF than those treated without EGF, suggesting that EGF may stimulate the proliferation of keratocytes in the early stage of wound healing.

Topics: Alkalies; Animals; Burns, Chemical; Cell Division; Cornea; Corneal Injuries; Epidermal Growth Factor; Eye Burns; Rabbits; Wound Healing

Previous in vivo studies evaluating the effects of growth factors on epithelial regeneration have used the scrape injury model in rabbit eyes. Since growth factors act principally on the mitotic activity of regenerating cells, the rapid wound closure rates following scrape injury may not adequately access the effects of these agents on epithelial repair. In this study, we evaluated the rates of wound healing following scrape (8.6 mm) and lamellar keratectomy (8.6 mm) injury in 25 albino rabbits. Eyes were left untreated or received daily application (two to three times) of (a) Tears Naturale II, (b) 50 mM Tris/NaCl, and (c) commercial vehicle for EGF. Eyes were evaluated daily by fluorescein staining with Ophthalmic Fluoro-Strips followed by clinical photography. The area of staining was quantitated by computer-assisted planimetry and rates calculated by linear regression analysis. Eyes receiving scrape injuries epithelialized by 3 days following surgery. Rates of wound closure in two separate groups (six eyes each) were 25.83 mm2/day (r = 0.96) and 29.56 mm2/day 9r = 0.97). Lamellar keratectomy injuries epithelialized by 7 to 8 days, which is substantially longer than that observed for scrape injuries. Rates of wound healing in two separate untreated groups (8 and 10 eyes) were 10.88 mm2/day (r = 0.95) and 9.00 mm2/day (r = 0.93), respectively, which were not significantly different. Analysis of variance comparing rates of wound closure indicated that lamellar keratectomy injuries heal at a significantly slower rate when compared to scrape injury (p less than 0.0005).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cell Movement; Cornea; Corneal Injuries; Corneal Stroma; Epidermal Growth Factor; Epithelium; Ophthalmic Solutions; Rabbits; Regeneration; Regression Analysis; Tromethamine; Wound Healing

The influence on human epidermal growth factor (hEGF) on the healing of a standardized corneal alkali wound was studied in the rabbit. The epithelial, stromal, and endothelial healing processes were followed separately for three weeks using quantitative methods. Epithelial and stromal healing was statistically significantly better with h-EGF (0.05 mg/ml) applied topically three times per day. However, hEGF induced considerable neovascularization, and some of the positive influence could be a result of the new vessel formation.

Topics: Administration, Topical; Alkalies; Animals; Cornea; Corneal Injuries; Corneal Stroma; Endothelium, Corneal; Epidermal Growth Factor; Epithelium; Neovascularization, Pathologic; Rabbits; Time Factors; Wound Healing

Epidermal growth factor (EGF) is known to promote corneal epithelial wound healing in experimental scrape and keratectomy models. We studied its efficacy in treating alkali-burned epithelial ulceration. EGF induced hyperplasia and cell proliferation to resurface the denuded and denatured corneal stroma, but it did not prevent recurrent erosions. A combination of EGF with fibronectin (Fn) was noted to enhance epithelial defect closure after alkali burns of the cornea and was seen to prevent recurrent erosions. Histopathologic examination of these corneas revealed marked leukocytic infiltration of the alkali-burned corneal stroma. To find ways to retard this inflammatory response, we studied the role of topical steroids and their efficacy when used with EGF, Fn, and laminin (Ln) in the management of alkali-burned corneas. Use of steroids decreased the incidence of recurrent erosions and corneal perforations. Histologically, steroids markedly decreased the leukocytic infiltration of stromal tissue, thereby retarding the collagenolysis. Topical steroids used with EGF and fibronectin were seen to promote epithelial wound closure and to prevent recurrent erosions in alkali-burned corneas. Combinations of EGF, fibronectin, and steroids may have a place in the treatment of clinical corneal alkali burns.

Topics: Adrenal Cortex Hormones; Animals; Cell Division; Corneal Injuries; Corneal Ulcer; Epidermal Growth Factor; Epithelium; Eye Burns; Fibronectins; Ophthalmic Solutions; Rabbits; Recurrence; Sodium Hydroxide; Wound Healing

Epidermal growth factor was injected intracamerally into the anterior chamber of the right eye of 9 cats. The central portion of the cornea in 8 of the 9 cats that had been cryoinjured. Effect of epidermal growth factor on the repair of endothelial cells in cats was evaluated by endothelial specular microscopy. Endothelial cell density and corneal thickness were studied quantitatively, as a measure of endothelial cell function. The repair process also was evaluated qualitatively by studying morphologic changes, developing as a result of reendothelialization and return to normal function. Seemingly, differences between rate of healing of cryoinjured eyes injected with epidermal growth factor and that in nontreated eyes were not significant (P = 0.86). The endothelial repair process was characterized by enlargement and migration of adjacent noninjured cells.

Topics: Animals; Cat Diseases; Cats; Cornea; Corneal Injuries; Endothelium, Corneal; Epidermal Growth Factor; Female; Male

The dose-dependent effect of epidermal growth factor (EGF) on the development of wound tensile strength following full-thickness corneal wounds was evaluated in 60 adult rabbits. One eye from each rabbit received a single 7-mm long corneal incision. After injury each rabbit was treated three times daily for 5 or 10 days with either EGF at 0.001 mg/ml (10 eyes), 0.01 mg/ml (10 eyes), 0.1 mg/ml (10 eyes), 1.0 mg/ml (15 eyes), or vehicle (15 eyes). The tensile strength of the wound was evaluated using a 5-mm wide strip of cornea mounted on a tensiometer. We found that EGF at 0.1 mg/ml and at 0.01 mg/ml increased wound strength by 100% at 5 days and by 60% at 10 days (P less than 0.05 and P less than 0.05). However, EGF at 0.001 mg/ml and 1.0 mg/ml appeared to have no effect on wound strength. Histologic examination of full-thickness wounds in a separate series showed an increase in wound fibroblastic response and a diminished fibrin clot at 5 days in rabbits treated with 0.1 mg/ml and 0.01 mg/ml. We conclude that EGF enhances the wound strength of full-thickness corneal wounds in a dose-dependent manner which may be explained in part by an increased fibroblastic response. Concentrations of EGF greater or less than an optimal dose may be less effective in enhancing corneal wound strength.

Topics: Administration, Topical; Animals; Cornea; Corneal Injuries; Dose-Response Relationship, Drug; Epidermal Growth Factor; Fibroblasts; Rabbits; Time Factors; Wound Healing

Regeneration of corneal epithelium following injury is essential for visual rehabilitation. A limited number of approaches are available for treating patients who fail to heal epithelial injuries adequately. The presence of specific receptors for epidermal growth factor (EGF) on epithelial cells suggests that this potent mitogen may play a role in normal epithelial wound healing. Topical application of biosynthetic human EGF significantly accelerated epithelial regeneration in primates following epikeratophakia surgery. Epidermal growth factor alone and with fibronectin accelerated epithelial regeneration of rabbits following mild alkali burns. Since prolonged exposure of cells to EGF is necessary to induce mitosis, the dynamics of EGF in the eye and with various lenses was studied. When applied in methylcellulose-based eye drops, 90% of the EGF was lost from tear film within 10 min, while a small amount (10%) remained associated with conjuctival tissue. Soft contact lenses or epikeratophakia lenticles took up substantial amounts of EGF (50 micrograms) and released 85% of the EGF within 24 h, with a half-life of 4 h in vitro. Epidermal growth factor did not diffuse through corneas or lenticles and did not promote epithelial downgrowth along sutures in primate corneas. These results suggest that biosynthetic growth factors may be useful in the treatment of some epithelial injuries.

Topics: Alkalies; Animals; Burns, Chemical; Contact Lenses, Hydrophilic; Cornea; Corneal Injuries; Corneal Transplantation; Epidermal Growth Factor; Epithelium; Eye Burns; Iodine Radioisotopes; Regeneration

We conducted a double-masked study to evaluate the effect of epidermal growth factor on epithelial wound healing and recurrent erosions in alkali-burned rabbit corneas. Epithelial wounds 10 mm in diameter healed completely under the influence of topical epidermal growth factor, whereas the control corneas did not resurface in the center. On reversal of treatment, the previously nonhealing epithelial defects healed when treated with topical epidermal growth factor eyedrops. Conversely, the epidermal growth factor-treated and resurfaced corneas developed epithelial defects when treatment was discontinued. Histopathologic examination disclosed hyperplastic epithelium growing over the damaged stroma laden with polymorphonuclear leukocytes when treated with epidermal growth factor eyedrops, but it did not adhere to the underlying tissue. Hydropic changes were seen intracellularly as well as between the epithelial cells and the stroma.

Topics: Alkalies; Animals; Burns, Chemical; Corneal Injuries; Double-Blind Method; Epidermal Growth Factor; Eye Burns; Kinetics; Rabbits; Time Factors; Wound Healing

Twenty pigmented rabbits weighing 2-3 kg were employed. Under operating microscope control, a Graefe knife was used to perform a 6-mm-long perforating incision in the central cornea. The operated eye which showed a stimulation unit at 4 ng X ml-1 on rat 3T3 cells in 10 rabbits was treated locally with sterile PBS, and on day 30 the rabbits were sacrificed and the operated eye enucleated. The central cornea was excised using a cutting template 10 X 3 mm, with the long axis perpendicular to the center of the experimental wound. The tensile strength was determined by measuring the force of rupture of the wounds using a dynamometer. The results indicate that epidermal growth factor significantly (p less than 0.001) increased corneal wound tensile strength after the first month of healing.

Topics: Animals; Cornea; Corneal Injuries; Epidermal Growth Factor; Rabbits; Tensile Strength; Wound Healing

Treatment of rabbit corneal wounds with topical corticosteroid retards both epithelial regeneration and healing of penetrating stromal wounds. Currently, no clinical agent is available which accelerates the rate of stromal wound healing. Epidermal growth factor (EGF, 0.5 mg ml-1), fibroblast growth factor (FGF, 20 micrograms ml-1), and insulin (0.5 mg ml-1) were tested for their ability to accelerate healing of totally penetrating wounds in rabbit corneas when the hormones were administered alone or in combination with dexamethasone (1 mg ml-1). After 5 days of treatment with eye drops, the tensile strengths of corneal wounds treated with EGF (54 +/- 4 g mm-1) or treated with EGF and dexamethasone (32 +/- 9 g mm-1) were significantly higher than the tensile strengths of corneal wounds treated with only saline vehicle (3 +/- 1 g mm-1) or dexamethasone (1 +/ 0 g mm-1) (P less than 0.001). The combination of dexamethasone with EGF significantly (P less than 0.025) reduced the strength of corneal wounds compared to treatment with EGF alone. Similarly, the tensile strength of corneal wounds after 5 days of insulin treatment alone (28 +/- 8 g mm-1) or in combination with dexamethasone (25 +/- 7 g mm-1) was significantly increased compared with saline- or dexamethasone-treated corneas (P less than 0.001). In the absence of dexamethasone, EGF increased the tensile strength of corneal wounds significantly better than insulin (P less than 0.01). However, when EGF or insulin were given in combination with dexamethasone there was no significant difference between the tensile strength produced by the peptide hormones. In comparison to the tensile strength of corneal wounds treated by EGF or insulin, treatment with FGF alone (5 +/- 4 g mm-1) or in combination with dexamethasone (2 +/- 1 g mm-1) produced poor wound healing. The in vitro actions of EGF or FGF alone or in combination with dexamethasone were tested for ability to stimulate [3H]-thymidine incorporation into pure cultures of human corneal fibroblasts (HCF) in defined culture medium. EGF (5 mM) or FGF (100 ng ml-1) alone stimulated [3H]-thymidine incorporation approximately 2.5-fold compared to control cultures, whereas in combination with dexamethasone (10 nM), the stimulatory action of FGF, but not EGF, was abolished. Dose-response curves indicated that HCF in culture were very sensitive to EGF, insulin, and FGF with maximum stimulation of [3H]-thymidine incorporation occurring at approximately 1 nM for EGF

Topics: Animals; Cells, Cultured; Cornea; Corneal Injuries; Dexamethasone; DNA; Dose-Response Relationship, Drug; Drug Synergism; Epidermal Growth Factor; Fibroblast Growth Factors; Fibroblasts; Humans; Insulin; Male; Rabbits; Tensile Strength; Wound Healing

Topics: Animals; Cornea; Corneal Injuries; Epidermal Growth Factor; Epithelium; Fibronectins; Rabbits; Regeneration; Wound Healing

The effects of the topical application and the intraperitoneal administration of epidermal growth factor (EGF) on the healing of epidermal wounds, and of partial-thickness scalds in rats and of corneal wounds in rabbits were evaluated. The parameters measured were the daily percentage body weight change, time to complete healing of the wounds, the evaporative water loss from the wound surface and the rate of new formation of epithelial cell layers on the epidermal and corneal wounds. Wounded animals treated with 0.9% NaCl were used as controls. After removal of the corneal epithelium the local application of EGF stimulated the regeneration process appreciably. No significant healing advantage from the treatment with EGF was observed, however, in epidermal wounds and partial-thickness scalds. The result does not warrant any clinical evaluation.

Topics: Animals; Body Water; Body Weight; Burns; Cell Division; Cornea; Corneal Injuries; Epidermal Growth Factor; Epidermis; Mice; Rabbits; Rats; Rats, Inbred Strains; Skin; Wound Healing

Topics: Animals; Cattle; Corneal Injuries; Endothelium; Epidermal Growth Factor; Fibroblasts; Growth Substances; Organ Culture Techniques; Peptides; Wound Healing

Epidermal growth factor (EGF) is a polypeptide hormone present in mammalian organs. In vivo, it shortens the time course of the corneal reepithelialization by stimulating a marked cell proliferation of the corneal epithelium. A further direct effect in vivo has been confirmed on human corneal epithelium and epidermis in culture. Tests in several nondystrophic diseases of the corneal epithelium confirmed the observations previously made in the rabbit that EGF accelerates the process of epithelial healing. The integrity of the corneal stroma is prejudicial for the maximum effect of the EGF, in the sense that the deeper the stroma is damaged, the less EGF acts. In herpetic lesions EGF is effective within 48 h only when the virus-affected area of the corneal epithelium has been scraped off. EGF is proposed as a new and efficacious agent for increasing the restorative process of the corneal epithelium in many nondystrophic diseases.

Topics: Adolescent; Adult; Aged; Animals; Child; Cornea; Corneal Diseases; Corneal Injuries; Epidermal Growth Factor; Epithelium; Female; Humans; Male; Middle Aged; Peptides; Rabbits; Wound Healing