Expression of HGF, KGF, EGF and receptor messenger RNAs following corneal epithelial wounding

Hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), epidermal growth factor (EGF), and their receptors have been associated with homeostasis and wound healing in the cornea. The purpose of this study was to examine the expression of the messenger RNAs for these growth factors and receptors in a wounded series of mouse corneas using in situ hybridization. In situ hybridization was performed with 3H-labeled riboprobes on unwounded corneas and corneas at 30 minutes, 4, 12, 24, 48 and 72 hr, and 7 days after epithelial scrape wounds in Balb/C mice. Qualitative and semi-quantitative analyses were performed. Expression of HGF, KGF and EGF mRNAs in keratocytes in the unwounded cornea was low. EGF mRNA was also expressed in unwounded corneal epithelium. Following wounding, however, these growth factor mRNAs were markedly upregulated in keratocytes. EGF mRNA expression in the epithelium appeared unaffected by wounding. At seven days after wounding and several days following closure of the epithelial defect, HGF mRNA and KGF mRNA were still expressed at higher levels in keratocytes compared with unwounded corneas. No difference in expression of HGF or KGF mRNAs between limbal, peripheral corneal, or central corneal keratocytes was noted in the unwounded cornea, KGF receptor mRNA was prominently expressed throughout the unwounded corneal epithelium. HGF receptor mRNA and EGF receptor mRNAs were expressed at low levels in unwounded cornea epithelium. Following scrape injury, expression of HGF receptor mRNA and KGF receptor mRNA were markedly upregulated in the corneal epithelium, while no significant increase in EGF receptor mRNA expression was noted. These studies suggest a prominent role for HGF and KGF in modulating corneal epithelial wound healing following injury. Less prominent changes in EGF mRNA and EGF receptor mRNA in the corneal epithelium following wounding may suggest that EGF has more of a role in homeostasis in the mouse corneal epithelium.     

Lacrimal gland HGF, KGF, and EGF mRNA levels increase after corneal epithelial wounding.

PURPOSE: To evaluate the effect of corneal epithelial wounding on lacrimal gland expression of hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), and epidermal growth factor (EGF) in the rabbit model. METHODS: Rabbits had corneal epithelial scrape injuries, and the lacrimal gland was removed at different times after wounding. HGF, KGF, and EGF mRNA expression was examined by quantitative RNase protection assay. HGF, KGF, and EGF proteins were detected in rabbit lacrimal tissue using immunoprecipitation and western blot analysis. RESULTS: HGF mRNA and EGF mRNA were significantly increased in rabbit lacrimal gland tissue within 8 hours after corneal epithelial injury. The increase in KGF mRNA expression was small and reached significance I clay after corneal injury. Lacrimal gland expression peaked at 3 days after wounding for each growth factor mRNA, the same day, on average, that the epithelial defect healed. After the peak increase in expression, there was a progressive decline in expression of each growth factor mRNA, but production was still increased compared with prewound levels. HGF protein, KGF protein, and EGF proteins were detected in rabbit lacrimal gland tissue. CONCLUSIONS: Levels of HGF, KGF, and EGF mRNAs increase in rabbit lacrimal gland tissue in response to corneal epithelial wounding. The results of this study are consistent with the existence of a cornea-nervous system-lacrimal gland regulatory loop modulating expression of these growth factor mRNAs. The lacrimal gland is a likely source of increased HGF and EGF proteins detected in tears in previous studies.     

The epidermal growth factor receptor (EGFR): role in corneal wound healing and homeostasis

To evaluate the role of the epidermal growth factor receptor (EGFR) in corneal epithelial wound healing, the effect of an EGFR inhibitor on epithelial cell proliferation and cell stratification during wound healing was investigated. From 3 days prior to wounding until wound healing was complete, rats were systemically treated with either an EGFR tyrosine kinase inhibitor (ZD1839) at 40 mg kg(-1) day(-1)or 80 mg kg(-1) day(-1), or with vehicle only (control). A single corneal wound was made in the center of 66 rat corneas, using a 6.0 mm glass tube wrapped in tissue paper soaked in n-heptanol. Subsequently, each wound was photographed and measured by a computer-assisted digitizer every 12 hr. To determine the number of cells in S phase, entire corneas were labelled with (3)H-thymidine and subjected to autoradiography at 0, 12, 24 and 48 hr after wounding. Epithelial thickness was also measured at these time points by microscopy. Epithelial wound healing was significantly and dose-dependently delayed following administration of ZD1839. At 24 hr after wounding, the number of S-phase cells in the limbal corneal epithelium was significantly lower in both the treated groups compared with the control group (P < 0.05). In the cornea before wounding (0 hr) and at 48 hr post-wounding, epithelial thickness was also significantly less in treated rats compared with controls (P < 0.05). These results indicate that EGFR inhibition affects epithelial cell proliferation and stratification during corneal epithelial wound healing and may play a role in maintaining normal corneal epithelial thickness.     

Normal corneal wound healing

Corneal wound healing is divided into several phases that, in reality are part of a continuous process, involving the corneal epithelium and stroma, corneal nerves, inflammatory cells, lacrimal glands. The layers of the cornea interact through autocrine and paracrine modulation, mediated by cytokines, growth factors, proteolytic enzymes. Corneal healing is affected by many factors, including: wound size and depth, causative agent, age, medication, preexisting diseases.     

Detection of transforming growth factor-alpha messenger RNA and protein in human corneal epithelial cells.

Human corneal epithelial cells are normally shed from the apical surface and replaced primarily by mitosis of basal cells. Growth factors may regulate this process, but the sources for the growth factors have not been fully established. One potential source for growth factors is tear fluid, and epidermal growth factor (EGF) has been detected in the lacrimal gland and in tears. However, the hydrophilic structure and size of growth factors such as EGF may limit penetration to basal layers of intact epithelium. It is possible that turnover of basal human corneal epithelial cells might be regulated by growth factors acting by an autocrine mechanism. To determine if human corneal epithelial cells synthesize a potential autocrine growth factor, the authors analyzed human corneal epithelial cells for transforming growth factor-alpha (TGF-alpha) messenger RNA and protein, a growth factor that is structurally related to EGF and binds to the EGF receptor. Radioimmunoassay of human corneal epithelial cell cultures detected substantial levels of immunoreactive TGF-alpha (3 ng/10(6) cells). Immunohistochemical staining of human corneas also revealed the presence of immunoreactive TGF-alpha in the corneal epithelium. Northern hybridization with a 32P-labeled complementary DNA probe for TGF-alpha generated a single intense band at 4.4 kilobases, indicating the presence of TGF-alpha messenger RNA in cultured human corneal epithelial cells. These results support the hypothesis that normal turnover of corneal epithelium is controlled by the autocrine production of growth factors, such as TGF-alpha. Growth factors present in tears may function primarily as exocrine factors to stimulate healing of epithelial injuries after the epithelial barrier has been damaged.     

Release of neuronotrophic factor from rabbit corneal epithelium during wound healing and nerve regeneration

Epithelial neuronotropic factor (ENF) is secreted by cultured epithelial cells of rabbit cornea and conjunctiva, and is active in promoting survival and inducing neurite outgrowth of cultured trigeminal neurons. This study evaluated the relation of ENF to corneal nerve regeneration utilizing a model of heptanol-induced epithelial wounding. The organ culture technique was used to collect ENF from the intact corneal epithelium, and a neuronal bioassay was utilized to quantify ENF. The results revealed no change in ENF secretion either during initial wound closure or after 1 week, when the epithelium had regenerated. However, ENF secretion was elevated 2.4 times in 2 weeks after wounding. Morphometric analysis of corneal nerves stained by gold chloride impregnation showed that the first sign of regeneration of intraepithelial nerves was observed after 2 weeks, and the normal pattern of epithelial neural density was re-established after 3 weeks. However, the neural density was still subnormal (35-47% less than the control) in the wounded epithelium up to 4 weeks after wounding. Thus it appears that a surge in ENF secretion occurred after epithelial regeneration but before nerve regeneration. The results suggest that ENF may mediate corneal nerve regeneration.     

Secretory immune system of rabbit ocular adnexa.

A distinct system of immunity in a variety of animals is located subjacent to epithelial surfaces and is typified by the predominance of immunoglobulin A (IgA) and secretory component (SC) in various external secretions, including tears. The present study examined normal rabbit lacrimal gland, conjunctiva, and cornea for the presence of immunoglobulin and SC. IgA-staining plasma cells predominated within lacrimal gland and conjunctival stroma, and SC was found in the epithelial cells of both these tissues but not within corneal epithelium. These observations are consistent with findings for other secretory sites in both rabbits and humans and establish lacrimal gland and conjunctiva as integral parts of the rabbit secretory immune system.     

Stromal-epithelial interactions in the cornea

Stromal-epithelial interactions are key determinants of corneal function. Bi-directional communications occur in a highly coordinated manner between these corneal tissues during normal development, homeostasis, and wound healing. The best characterized stromal to epithelial interactions in the cornea are mediated by the classical paracrine mediators hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF). HGF and KGF are produced by the keratocytes to regulate proliferation, motility, differentiation, and possibly other functions, of epithelial cells. Other cytokines produced by keratocytes may also contribute to these interactions. Epithelial to stromal interactions are mediated by cytokines, such as interleukin-1 (IL-1) and soluble Fas ligand, that are released by corneal epithelial cells in response to injury. Other, yet to be identified, cytokine systems may be released from the unwounded corneal epithelium to regulate keratocyte viability and function. IL-1 appears to be a master regulator of corneal wound healing that modulates functions such as matrix metalloproteinase production, HGF and KGF production, and apoptosis of keratocyte cells following injury. The Fas/Fas ligand system has been shown to contribute to the immune privileged status of the cornea. However, this cytokine-receptor system probably also modulates corneal cell apoptosis following infection by viruses such as herpes simplex and wounding. Pharmacologic control of stromal-epithelial interactions appears to offer the potential to regulate corneal wound healing and, possibly, treat corneal diseases in which these interactions have a central role.     

Connective tissue growth factor in tear film of the horse: detection,identification and origin

Background Healing of corneal ulcers in horses is often associated with profound corneal stromal fibrosis and scar formation resulting in visual impairment. Connective tissue growth factor (CTGF) is a fibrogenic cytokine involved in wound healing and scarring. The purpose of this study was to determine whether CTGF was present in the tear fluid of normal horse eyes and the eyes of horses with corneal ulcers in order to evaluate the role of CTGF in corneal wound healing and corneal scar formation.Methods Tear fluid samples were collected from 65 eyes of 44 horses; 32 samples from normal eyes, 21 samples from eyes with corneal ulceration, and 12 samples from the unaffected contralateral eyes of horses with ulcers. CTGF levels in the tears were determined by enzyme immunoassay using goat IgG against human CTGF. Antigenetic similarity of human and horse CTGF was established in a bio-equivalence assay. The identity of horse CTGF was confirmed by western blot. Lacrimal and nictitating membrane glands were investigated by immunohistochemistry in the attempt to clarify the origin of tear fluid CTGF.Results CTGF was detected in tear film of 23 normal unaffected eyes (72%) and 8 normal contralateral eyes (67%), with the mean CTGF levels (± SEM) being 51.5±19.2 and 13.4±3.9 ng/ml respectively. CTGF was found in 8 eyes with corneal ulcers (38%) with the mean CTGF concentration of 26.3±14.8 ng/ml. Western blot identified the protein detected as CTGF. The identification of CTGF in lacrimal glands suggests a major role of these glands in the presence of CTGF in tears.Conclusions CTGF is present in horse tear fluid and derives, at least partly, from the lacrimal gland. Equine CTGF has strong antigenic similarity with human CTGF. Corneal disease leads to a decrease of CTGF concentrations in tears. The possible role of CTGF in the healing process of ocular surface requires further investigation.     

The corneal wound healing response: cytokine-mediated interaction of the epithelium, stroma, and inflammatory cells

The corneal wound healing cascade is complex and involves stromal-epithelial and stromal-epithelial-immune interactions mediated by cytokines. Interleukin-1 appears to be a master modulator of many of the events involved in this cascade. Keratocyte apoptosis is the earliest stromal event noted following epithelial injury and remains a likely target for modulation of the overall wound healing response. Other processes such as epithelial mitosis and migration, stromal cell necrosis, keratocyte proliferation, myofibroblast generation, collagen deposition, and inflammatory cell infiltration contribute to the wound healing cascade and are also likely modulated by cytokines derived from corneal cells, the lacrimal gland, and possibly immune cells. Many questions remain regarding the origin and fate of different cell types that contribute to stromal wound healing. Over a period of months to years the cornea returns to a state similar to that found in the unwounded normal cornea.     

The contribution of blood flow by the anterior ciliary arteries to the anterior segment in the primate eye

The rate and mode of corneal wound healing in severely diabetic rats were studied by light microscopy and scanning electron microscopy. Diabetes mellitus was induced in 52 rats by alloxan injection, and 52 nondiabetic rats were used as controls. After 3 weeks, a nonpenetrating razor-blade wound was made in the central cornea of both eyes in 48 diabetic and 48 normal rats. The incision passed through the epithelium and into the stroma. The effects of diabetes on the unwounded cornea were observed by comparison with corneas from eight unwounded rats (four diabetic and four normal). Whole corneas from wounded diabetic and normal rats were studied at 0, 1, 3, 6, 12 and 24 hr and at 2–7 days after wounding. The rate and mode of healing were not found to differ between diabetics and normals. The surfaces of corneal wounds in both groups appeared to be completely healed and indistinguishable from the surrounding unwounded epithelium after 24 hr. The epithelial cells involved in the initial healing process were derived primarily from the layer of wing cells which progressed across the wound close to the connective-tissue base. Only in the final stages of healing, after the wound had been filled by the deeper epithelial cells, did superficial epithelial cells migrate. There appeared to be more exfoliating superficial epithelial cells over the entire cornea in diabetic rats than in normals. Because the healing of central corneal incisions occurs initially and primarily by sliding of the deeper epithelial cells, and because the diabetic condition appears to be associated with increased exfoliation of surface cells, the healing of central incisions may be less affected by diabetes than the healing of defects of the whole corneal surface, where the superficial epithelial cells have been reported to be the main migratory cells in the initial healing process and where healing in diabetics is delayed.     

Growth factors in the tear film: role in tissue maintenance, wound healing, and ocular pathology

Numerous biologically active growth factors are secreted by the lacrimal gland and distributed via the tears over the ocular surface, where they affect cellular proliferation, migration, differentiation, and survival. The role of growth factors and their receptors in maintenance of tissue homeostasis and wound healing continues to be elucidated, and the effect of growth factor imbalances in ocular surface diseases is just beginning to be understood. For instance, in eyes with ocular surface diseases, including conjunctivitis, corneal erosion, keratitis, and corneal ulcers, epidermal growth factor release rates have been shown to be significantly lower than in normal eyes during reflex tearing. Future research into the mechanisms of dry eye disease will focus on reasons for decreased tear and growth factor production in the neuronal reflex loop or the acinar lacrimal gland cells. Animal models to test therapeutic approaches must be developed.     

Growth factor, cytokine and protease interactions during corneal wound healing

Healing of corneal injuries is an exceptionally complex process involving the integrated actions of multiple growth factors, cytokines, and proteases produced by epithelial cells, stromal keratocytes, inflammatory cells, and lacrimal gland cells. Following corneal injury, basal epithelial cells migrate and proliferate in response to chemotactic cytokines and mitogenic growth factors, including epidermal growth factor and keratinocyte growth factor. Simultaneously, keratocytes adjacent to the injured area undergo apoptosis under the Fas/Fas ligand system, while more distant keratocytes transform into activated fibroblasts and migrate into the wound, where they begin synthesizing new extracellular matrix components that form the scar tissue under the dominant influence of the TGFb/ CTGF system. Epithelial cells and activated stromal fibroblasts also secrete growth factors and cytokines that have paracrine and autocrine functions. Corneal repair proceeds for the next several weeks to months, during which time the gene expression profile slowly returns to the pre-injury pattern and the provisional scar matrix slowly remodels by actions of matrix metalloproteinases. While minor epithelial injuries heal by regeneration of normal architecture, large stromal injuries heal by repair with irregular scar tissue that impairs the optical properties of the cornea.Also, if the integrated regulation of the wound healing process is interrupted at any point, the wound fails to heal properly and a corneal ulcer develops. Better understanding of the cellular and molecular changes that occur during repair of corneal wounds will provide the opportunity to design agents that selectively modulate key phases of corneal wound healing, resulting in scars that more closely resemble normal corneal architecture.     

Regeneration of rabbit cornea following excimer laser photorefractive keratectomy: a study on gap junctions, epithelial junctions and epidermal growth factor receptor expression in correlation with cell proliferation

Corneal wound repair was investigated in rabbits following excimer laser ablation of a 6 mm diameter and 90 microm deep disc. In the healing process particular attention was focused on the epithelium where gap junction expression and the rearrangement of desmosomes and hemidesmosomes were correlated with cell proliferation and epidermal growth factor receptor expression. Immunofluorescence-based confocal laser scanning microscopy, semithin resin section morphology and electron microscopy were utilized. In resting cornea two isotypes of gap junctions, confined to different regions in the same basal epithelial cells, were detected. Particulate connexin43 (alpha1) immunostaining was concentrated on the apical while the connexin26 type (beta2) in the baso-lateral cell membranes. This is the first report of connexin26 in the cornea. Connexin43 was found also in corneal keratocytes and endothelial cell. Since the two connexins do not form functioning heteromeric channels and have selective permeabilities they may serve alternative pathways for direct cell-cell communication in the basal cell layer. During regeneration both connexins were expressed throughout the corneal epithelium including the migrating cells. They also showed transient up-regulation 24 hr after wounding in the form of overlapping relocation to the upper cell layers. At this time, basal epithelial cells at the limbal region, adjacent to the wound and those migrating over the wounded area all expressed membrane bound epidermal growth factor receptor and they were highly proliferating. In conclusion, like in other stratified epithelia connexin26 is also expressed in the cornea. Transient up-regulation and relocation of connexins within the regenerating epithelium may reflect the involvement of direct cell-cell communication in corneal wound healing. Mitotic activity in the migrating corneal epithelial cells is also a novel finding which is probably the sign of the excessive demand for new epithelial cells in larger wounds not met alone by the proliferating limbal stock.     

Detection of surfactant proteins A and D in human tear fluid and the human lacrimal system

PURPOSE: To evaluate the expression and presence of surfactant protein (SP) A and SP-D in the lacrimal apparatus, at the ocular surface, and in tears in healthy and pathologic states. METHODS: Expression of mRNA for SP-A and SP-D was analyzed by RT-PCR in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal ducts as well as in a spontaneously immortalized conjunctival epithelial cell line (HCjE; IOBA-NHC) and a SV40-transfected cornea epithelial cell line (HCE). Deposition of SP-A and SP-D was determined by Western blot, dot blot, and immunohistochemistry in healthy tissues, in tears, aqueous humor, and in sections of different corneal abnormalities (keratoconus, herpetic keratitis, and Staphylococcus aureus-based ulceration). Cell lines were stimulated with different cytokines and bacterial components and were analyzed for the production of SP-A and SP-D by immunohistochemistry. RESULTS: The presence of SP-A and SP-D on mRNA and protein levels was evidenced in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal duct samples. Moreover, both proteins were present in tears but were absent in aqueous humor. Immunohistochemistry revealed the production of both peptides by acinar epithelial cells of the lacrimal gland and epithelial cells of the conjunctiva and nasolacrimal ducts, whereas goblet cells revealed no reactivity. Healthy cornea revealed weak reactivity on epithelial surface cells only. In contrast, SP-A and SP-D revealed strong reactivity in patients with herpetic keratitis and corneal ulceration surrounding lesions and in several immigrated defense cells. Reactivity in corneal epithelium and endothelium was also seen in patients with keratoconus. Cell culture experiments revealed that SP-A and SP-D are produced by both epithelial cell lines without and after stimulation with cytokines and bacterial components. CONCLUSIONS: These results show that SP-A, in addition to SP-D, is a peptide of the tear film. Based on the known direct and indirect antimicrobial effects of collectins, the surfactant-associated proteins A and D seem to be involved in several ocular surface diseases.     

角膜伤口愈合机制对准分子激光角膜屈光手术的启示

角膜伤口愈合反应是由细胞因子、生长因子、趋化因子介导的复杂级联反应。角膜上皮细胞和基底膜在这一过程中起了重要的作用,而这二者在准分子激光角膜屈光手术中常常被损伤。因此,深刻理解角膜伤口愈合过程以及并发症产生的原因,对提高准分子激光角膜屈光手术的有效性和安全性具有重要意义。就角膜伤口愈合过程及其机制进行综述。     

Absent meibomian glands in the ectrodactyly, ectodermal dysplasia, cleft lip-palate syndrome

A 27-year-old woman with the syndrome characterized by ectrodactyly, ectodermal dysplasia, and cleft lip-palate had an absent lacrimal punctum in each eye, with signs and symptoms of nasolacrimal obstruction during childhood. Examination disclosed bilateral corneal vascularization and opacification, with diffuse superficial punctate staining of the ocular surface epithelium by fluorescein, an instantaneous tear film break-up time, and normal Schirmer tear measurements. A full-thickness biopsy specimen of the eyelid confirmed the absence of meibomian glands that had been suspected because of absent meibomian gland orifices and secretions. The total absence of meibomian gland secretions in this patient may be a primary feature of this case and may contribute to a lipid-deficient and unstable tear film with resultant desiccation and destruction of the ocular surface epithelium. Breakdown of the corneal epithelium in association with obstruction and infection of the nasolacrimal system may be a particularly disastrous combination for the cornea that resulted in the recurrent, severe bacterial corneal ulcers found in our patient.