Human idiopathic epiretinal membranes express NGF and NGF receptors

PURPOSE: Glial cells and fibroblasts (FBs) play a key role in epiretinal membrane (ERM) development and progression. Myofibroblasts (myoFBs), arising from these cells, can lead to the hypertrophic scars and tissue contraction observed in ERMs. Nerve growth factor (NGF) and transforming growth factor-beta1 (TGF-beta1) play a crucial role in FB activities. Therefore, the authors evaluated myoFBs in ERMs and NGF, trkA(NGFR and p75(NTR) expression, as well as TGF-beta1/TGF-betaRII levels in both ERMs and vitreous. METHODS: Eight idiopathic ERMs and vitreous were obtained from patients at the time of vitrectomy for macular pucker. Ten control vitreous were from donors. Biochemical and molecular analyses were performed to identify alpha-smooth muscle actin (alpha-SMA, a defined myoFB marker), NGF, trkA(NGFR)/p75(NTR), and TGF-beta1/TGF-betaRII. RESULTS: Every idiopathic ERM displayed alpha-SMA positive myoFBs, expressing NGF, trkA(NGFR), and p75(NTR). ERM vitreous showed a significant decrease in NGF protein coupled with a TGF-beta1 increase. In addition, vitreous cells showed an increase in trkA(NGFR)/p75(NTR) mRNA associated with a decrease in TGF-betaRII mRNA. CONCLUSIONS: Idiopathic ERMs were characterized by myoFBs. The expression of NGF, trkA, and p75 in local myoFBs associated with changes in ERM vitreous NGF suggests an involvement of NGF, as previously reported for TGF-beta1, in the evolution and myoFB-mediated contractile activity of ERMs.     

Relation between corneal haze and transforming growth factor-beta1 after photorefractive keratectomy and laser in situ keratomileusis.

PURPOSE: To investigate the relation between corneal haze formation and transforming growth factor-beta (TGF-beta) after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK). SETTING: Department of Ophthalmology, University of Tokyo Graduate School of Medicine, Tokyo, Japan. METHODS: White rabbits were divided into 4 groups, with each group receiving 1 of the following surgeries: manual epithelial abrasion, PRK, lamellar keratotomy, or LASIK. The degree of corneal haze was quantitatively analyzed by measuring the light scattering intensity of corneas before and 4 and 12 weeks after surgery. The expression of type IV collagen and TGF-beta1 in the corneas at baseline and at 4 weeks was examined immunohistochemically. RESULTS: The light scattering intensity was significantly greater 4 and 10 weeks after PRK. In contrast, epithelial abrasion, lamellar keratotomy, and LASIK did not influence the light scattering intensity of the corneas. Type IV collagen was detected in the basal lamina of the corneal epithelium and in Descement's membrane in the normal cornea. After epithelial abrasion, there was no change in the distribution of type IV collagen. Four weeks after PRK, the expression of type IV collagen was detected in the subepithelial layer of the laser-ablated area. Four weeks after lamellar keratotomy, type IV collagen was linearly and fragmentarily detected in the corneal stroma. Four weeks after LASIK, type IV collagen was linearly and continuously detected in the corneal stroma and was detected slightly in the subepithelial region of the laser-ablated area. In the normal corneas, the expression of TGF-beta1 was not detected in the keratocytes. Four weeks after PRK, the expression of TGF-beta1 increased in the keratocytes that proliferated in the subepithelial fibrous layer. In contrast, epithelial abrasion, lamellar keratotomy, and LASIK did not change the expression pattern of TGF-beta1 in the keratocytes. CONCLUSION: The multiplier effect of epithelial abrasion and excimer laser ablation in PRK may increase the expression of TGF-beta1 in keratocytes and induce corneal haze.     

Corneal cells: chatty in development,homeostasis, wound healing,and disease

PURPOSE: To provide an overview of cell-cell interactions in the cornea that have a critical role in corneal development, homeostasis, wound healing, and disease. DESIGN: Review. METHODS: Review of the literature. RESULTS; Cell-cell interactions make critical contributions to development, homeostasis, and wound healing in the cornea. Many of these interactions are mediated by cytokines, growth factors, and chemokines. The best characterized are stromal-epithelial interactions between epithelial cells and stromal cells such as keratocytes, keratoblasts, and myofibroblasts. However, interactions also occur between corneal nerves and epithelial cells and between corneal cells (epithelial cells and stromal cells) and corneal immune cells. Although investigations are limited, it is likely that there are interactions between corneal endothelial cells and keratocytes in the posterior stroma. CONCLUSIONS: Cellular communications in the cornea are critical during development, homeostasis, and wound healing. Disorders of cellular communication likely contribute to many corneal diseases.     

Nerve growth factor and corneal wound healing in dogs

Nerve growth factor in the tear film and corneal epithelium is hypothesized to play an important role in ocular surface maintenance and corneal wound healing. The purpose of this study was to determine the expression of nerve growth factor and its high affinity (trkA) receptor in tears, cornea, and lacrimal glands of normal dogs, the modulation of nerve growth factor and its trkA receptor during corneal wound healing, and the effect of topical nerve growth factor application on canine corneal epithelial wound healing. In the first of three experiments, the nerve growth factor content of tears, corneal epithelium, lacrimal gland, and 3rd eyelid gland was determined in normal dogs by enzyme-linked immunosorbent assay and the expression of nerve growth factor and its trkA receptor were evaluated in the cornea and lacrimal glands by immunohistochemistry. In a second experiment, unilateral corneal epithelial defects were created, and tissues were evaluated for changes in nerve growth factor or trkA expression for 1 week. In a third experiment, bilateral corneal epithelial defects were created and the right eyes in each animal were treated 4 times daily with either recombinant human nerve growth factor, murine nerve growth factor, or nerve growth factor-blocking antibody. The results of this study showed that nerve growth factor levels in normal dog tears, corneal epithelium, third eyelid gland and lacrimal gland were 15.4+/-4.6 ng ml(-1), 33.5+/-12.3, 52.4+/-17.4 and 48.8+/-9.4 ng g(-1), respectively. NGF and trkA receptors were identified by immunohistochemistry in all tissues examined. After unilateral corneal wounding, nerve growth factor concentration increased in the tears bilaterally for 3 days, especially in the wounded eye, and then returned to pre-wounding values. Nerve growth factor content, and immunohistochemical staining for nerve growth factor and trkA, increased significantly in the ipsilateral cornea epithelium following unilateral wounding. Nerve growth factor concentrations in lacrimal and third eyelid glands also increased bilaterally (p<0.01) after unilateral wounding. Time to wound closure and rate of epithelial migration did not differ significantly between nerve growth factor-treated, nerve growth factor antibody-treated, and control eyes. In conclusion, nerve growth factor is present under resting physiologic conditions in normal canine tears, and nerve growth factor and its trkA receptor are present under resting conditions in normal canine corneal epithelium, lacrimal gland and third eyelid gland. Nerve growth factor is elevated in the tears, cornea, and lacrimal glands after corneal epithelial wounding; however, topical application of nerve growth factor, or its blocking antibody does not modulate corneal wound healing in the normal dog eye.     

Expression of VSX1 in human corneal keratocytes during differentiation into myofibroblasts in response to wound healing

PURPOSE: To characterize the expression of the visual system homeobox gene (VSX1) in human corneal keratocytes both in vitro and in vivo. METHODS: The expression of VSX1 was evaluated through semiquantitative RT-PCR, immunofluorescence and in situ hybridization both in corneas (either freshly obtained or wounded) and in collagenase/hyaluronidase-isolated keratocytes grown in the absence or presence of serum to promote keratocyte-to-myofibroblast differentiation. RESULTS: Quiescent or resting keratocytes normally residing in the corneal stroma or cultured in vitro in the absence of serum did not express VSX1. In wounded corneas or when cultured in the presence of serum to mimic wound-healing responses, keratocytes underwent fibroblastic transformation (with appearance of alpha-SMA and disappearance of CD-34 and keratocan signals) and started expressing VSX1. CONCLUSIONS: The results show that VSX1 is expressed in vitro and in vivo during human corneal wound healing, a process in which differentiation of corneal keratocytes into myofibroblasts occurs. These data may help to elucidate the role of VSX1 in cornea physiology suggesting a potential involvement in cornea-related diseases such as keratoconus.     

Thy-1 distinguishes human corneal fibroblasts and myofibroblasts from keratocytes

After corneal injury, keratocytes become activated and transform into repair phenotypes-corneal fibroblasts or myofibroblasts, however, these important cells are difficult to identify histologically, compromising studies of stromal wound healing. Recent studies indicate that expression of the cell surface protein, Thy-1, is induced in fibroblast populations associated with wound healing and fibrosis in other tissues. We investigated whether keratocyte transformation to either repair-associated phenotype induced Thy-1 expression. Human corneal keratocytes were isolated by collagenase digestion. The cells were either processed immediately (i.e. freshly isolated keratocytes) or were cultured in the presence of 10% fetal bovine serum or transforming growth factor-beta to induce transformation to the corneal fibroblast and myofibroblast phenotypes, respectively. Thy-1 mRNA and protein expression by freshly isolated keratocytes and corneal fibroblasts were assessed by RT-PCR and Western blotting. mRNA also was extracted from the whole intact stroma and assessed by RT-PCR. Thy-1 was localised immunocytochemically in cultured human corneal fibroblasts, myofibroblasts, and in keratocytes in normal human corneal tissue sections. Thy-1 mRNA and protein were detectable in cultured human corneal fibroblasts, but not freshly isolated keratocytes. Whole uninjured stroma showed no detectable Thy-1 mRNA expression. Cultured human corneal fibroblasts and myofibroblasts both labelled for Thy-1, but keratocytes in the stroma of normal human cornea did not. We conclude that Thy-1 expression is induced by transformation of keratocytes to corneal fibroblasts and myofibroblasts, suggesting a potential functional role for Thy-1 in stromal wound healing and providing a surface marker to distinguish the normal keratocyte from its repair phenotypes.     

Involvement of insulin-like growth factor-I and insulin-like growth factor binding protein-3 in corneal fibroblasts during corneal wound healing

PURPOSE: The involvement of downstream messengers of transforming growth factor (TGF)-beta in the differentiation of corneal fibroblasts into myofibroblasts was investigated. The effects of insulin-like growth factor (IGF)-I and insulin-like growth factor binding protein (IGFBP)-3 upregulated by TGF-beta were examined in human corneal fibroblasts, and the possible involvement of IGF axis components in corneal wound healing was assessed in a mouse model. METHODS: Human corneal fibroblasts were incubated with TGF-beta2 or IGF-I, to investigate IGF-I, IGF-II, IGFBP-3, type I collagen, and alpha-smooth muscle actin (alpha-SMA) mRNA, as well as IGFBP-3 protein expression, during myofibroblast differentiation. DNA synthesis was evaluated with a 5-bromo-2'-deoxyuridine (BrdU) incorporation assay. IGFBP-3 mRNA expression, protein expression, and immunolocalization were investigated in mouse corneas after photorefractive keratectomy (PRK). RESULTS: TGF-beta2 treatment induced expression of IGF-I and IGFBP-3 mRNA and of IGFBP-3 protein in human corneal fibroblasts. TGF-beta2 and IGF-I both stimulated expression of type I collagen. TGF-beta2 but not IGF-I potently stimulated alpha-SMA mRNA expression. IGF-I potently stimulated basal DNA synthesis, whereas IGFBP-3 inhibited it. IGF-I potently stimulated proliferation of TGF-beta2-activated myofibroblasts without reversing the activated fibrogenic phenotype, whereas IGFBP-3 suppressed IGF-I-induced proliferation of corneal fibroblasts. IGFBP-3 mRNA and protein increased in mouse corneas soon after PRK, when in vivo immunostaining of the corneas showed expression of IGFBP-3 in the deep layer of the corneal stroma. CONCLUSIONS: These results suggest that during corneal wound healing, TGF-beta stimulates IGF axis components, whereas IGFBP-3 may modulate IGF-I-induced myofibroblast proliferation to suppress corneal mesenchymal overgrowth.     

Expression and function of fibroblast growth factor-inducible 14 in human corneal myofibroblasts

The interaction of fibroblast growth factor-inducible 14 (Fn14) and, its ligand tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is known to be important in wound healing of tissues. However, to our knowledge, expression and function of Fn14 in corneal myofibroblasts, which have a crucial role in wound healing of corneal stroma, has not been investigated. In this study, we investigated the expression and function of Fn14 in corneal myofibroblasts. Expression of Fn14 protein was assessed by flow cytometry. Corneal myofibroblasts showed strong expression of Fn14 protein, while keratocytes did not. TGF-β₁ promoted the differentiation of keratocytes into corneal myofibroblasts, and induced Fn14 expression. These data reveal that keratocytes phenotype determines the level of Fn14 expression. ELISA was used to detect chemokines and matrix metalloproteinases in the supernatant of corneal myofibroblasts cultured with or without stimulation by TWEAK and/or TGF-β₁. TWEAK increased the production of IL-8, MCP-1, and RANTES by corneal myofibroblasts via Fn14. TGF-β₁ augmented the TWEAK-induced production of these chemokines. TWEAK also increased the production of MMP-1 and -3 by corneal myofibroblasts via Fn14, while TGF-β₁ inhibited this effect of TWEAK on MMP production. TWEAK-induced phosphorylation of NF-κB and MAP kinase in corneal myofibroblasts. Furthermore, TWEAK partially inhibited the differentiation of keratocytes into corneal myofibroblasts promoted by TGF-β₁. These data suggest that the Fn14/TWEAK system may have several roles in wound healing by corneal myofibroblasts. In the future, modulation of the TWEAK/Fn14 system may become a novel approach for control corneal wound healing.     

Distribution of epidermal growth factor (EGF) receptors in rabbit corneal epithelial cells, keratocytes and endothelial cells, and the changes induced by transforming growth factor-beta 1.

Three kinds of rabbit corneal cells (epithelial cells, keratocytes and endothelial cells) were cultured, and the growth promoting effects of epidermal growth factor (EGF) were examined in these cells. It was found that the sensitivity of the epithelial cells to EGF was the highest, that of the endothelial cells was a little lower, and that of the keratocytes was the lowest. Transforming growth factor-beta 1 (TGF-beta 1) did not influence growth of the three kinds of corneal cells. It was found that TGF-beta 1 enhanced the growth promoting effect of EGF in the keratocytes, but not in the epithelial and endothelial cells. EGF receptors in the corneal cells were labelled with [125I]EGF and analysed by Scatchard plot. In the epithelial and endothelial cells, both high and low affinity receptors were found, while the keratocytes had only low affinity receptor. In the epithelial cells, the number and the association constant of the high affinity receptors were, respectively, 2.79 x 10(4) per cell and 0.034 nM, and those of the low affinity receptors were, respectively, 13.4 x 10(4) per cell and 0.700 nM. In the endothelial cells, the number and the association constant of the high affinity receptors were 1.27 x 10(4) per cell and 0.086 nM, respectively, and those of the low affinity receptors were 8.91 x 10(4) per cell and 1.536 nM. In the keratocytes, the number of receptors and the association constant were 9.49 x 10(4) per cell and 1.535 nM, respectively. The corneal cells were treated with TGF-beta 1 for 24 hr and its influence on EGF receptors was examined. The results showed that TGF-beta 1 induced the high affinity receptors in the keratocytes, although TGF-beta 1 did not influence EGF receptors in the epithelial and endothelial cells. The number of high affinity receptors in keratocytes treated with TGF-beta 1 was 1.02 x 10(4) per cell and the association constant was 0.171 nM (this was approximately tenfold higher than that of the receptor of keratocytes not treated with TGF-beta 1).     

Expression of transforming growth factor-beta in wound healing of vitamin A-deficient rat corneas

Transforming growth factor-beta (TGF-beta) is a multifunctional peptide that modulates cell proliferation and differentiation in many cell types and accelerates tissue repair response. In this study, expression of TGF-beta was investigated immunohistochemically in the healing of central 3 mm epithelial wounds of vitamin A-deficient (A-) rat and pair-fed controls. In control rat corneas, a positive reaction to TGF-beta was not evident during wound healing. In A- rat corneas at 4 hr post-abrasion, acute inflammatory cells showing high positivity to TGF-beta appeared in the peripheral stroma and gradually spread to the central cornea. By 24 hr, these cells accumulated and formed a pseudomembrane in the epithelial defect, which also showed an intense positivity to TGF-beta, suggesting that the peptide participates even in the acute inflammatory response. From about 16 hr post-injury, many fibroblasts revealing intense positivity to TGF-beta infiltrated the entire stroma and were part of the healing process. Reepithelialization occurred over the pseudomembrane and was completed by 48 hr. The central cornea showed remodeling of collagen structure and neovascularization. Fibroblasts containing TGF-beta were seen in the stroma, indicating that TGF-beta plays an important role in corneal wound healing. Indeed, in the absence of vitamin A, a common modulator of cell differentiation, TGF-beta may play a more important role in wound healing than in the normal state.     

Comparison of wound healing after photorefractive keratectomy and laser in situ keratomileusis in rabbits.

PURPOSE: To evaluate and compare the corneal wound-healing process after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK). SETTING: Kangnam St. Mary's Hospital, Seoul, Korea. METHODS: Two surgical procedures, PRK with the VISX Star excimer laser and LASIK with a MicroTech microkeratome, were performed in 24 rabbit eyes. In the PRK group (n = 12 eyes), the rabbit cornea was treated with a 20 microns ablation. In the LASIK group (n = 12 eyes), a 20 microns laser ablation was performed after a 150 microns thick hinged corneal flap had been made. During both procedures, dichlorotriazinyl aminofluorescien (DTAF) dye was applied to the ablated stromal bed; in the LASIK group, the stromal side of the corneal flap was also stained with DTAF to differentiate regenerated collagen from normal stromal tissue. Corneal wound healing was evaluated postoperatively at 1, 4, 8, and 12 weeks using light, electron, and fluorescence microscopy. The amount of regenerated stromal tissue and the number of keratocytes were analyzed by an image-analysis system. RESULTS: In the PRK group, epithelial migration and regeneration were observed in the ablated area without any stromal regeneration 1 week postoperatively. However, newly regenerated, irregularly arranged stromal collagen, with epithelial hyperplasia in the ablated area, was observed 4 to 12 weeks postoperatively by light and fluorescence microscopy. The number of keratocytes in the surgical area was also increased. In ultrastructural observation using an electron microscope, the shape of keratocytes in the ablated area was changed, and the number of rough and smooth endoplasmic reticuli, ribosomes, mitochondria, and electron-dense vesicles in the cytoplasm were increased, suggesting that the cells were activated. In the LASIK group, there was no observed regenerated collagen between the corneal flap and the ablated stromal bed except in the wound margin. Lamellated, parallel collagen fibers in the cornealstroma were not disturbed. However, in the wound margin, corneal epithelial ingrowth between the flap and the stromal bed was observed, as was some regenerated stromal tissue. The amount of regenerated stromal tissue and the number of keratocytes in the wound area were statistically smaller than those in the PRK group (P < .05). Observation by electron microscopy showed no activated keratocytes, unlike in the PRK group. The collagen fibers in the wound area were parallel. CONCLUSION: Stromal wound healing in the LASIK group was minimal compared with that in the PRK group, except in the wound margin. These results may support the clinical findings of less corneal haze in the human cornea after LASIK.     

Hepatocyte growth factor and keratinocyte growth factor regulation of epithelial and stromal corneal wound healing

PURPOSE: To investigate the effects of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) on early wound healing in the corneal epithelium and stroma. SETTING: Cell and Molecular Biology Unit, Department of Optometry and Vision Sciences, Cardiff University, and the Cardiff Institute of Tissue Engineering and Repair, Cardiff, United Kingdom. METHODS: Corneal keratocyte cell cultures and wounded corneal organ cultures (both maintained in serum-free conditions) were treated with 0.1 to 100 ng/mL of HGF or KGF for up to 5 days. Cell cultures were assessed for proliferation, migration, and differentiation into myofibroblasts. Organ cultures were used to evaluate the effect of HGF and KGF on reepithelialization following a wound, epithelial morphology and stratification, keratocyte numbers directly beneath the wounded area, and differentiation into myofibroblasts. RESULTS: The 2 growth factors had opposite effects on the rate of reepithelialization, with HGF delaying and KGF accelerating epithelial coverage of the wound. Morphologic assessment showed that both growth factors affected the stratification and differentiation of the epithelium. Both factors stimulated proliferation of keratocytes in serum-free cell culture, although neither induced the appearance of myofibroblasts. This was in contrast to wounded organ cultures treated with 100 ng/mL HGF, in which large numbers of myofibroblasts were observed under the wound. Control corneas and those receiving KGF contained very few myofibroblasts. Keratocyte repopulation of the denuded area under the wound was enhanced in the presence of HGF but decreased in response to KGF. CONCLUSIONS: Hepatocyte growth factor and KGF appeared to have potent and often opposite effects on epithelial and stromal cells following a wound. Hepatocyte growth factor was more detrimental than KGF, resulting in an aberrant epithelium and mass differentiation of keratocytes into myofibroblasts. Inhibition of HGF may be an appropriate therapeutic intervention in the case of persistent epithelial defects and to prevent fibrosis following a corneal stromal wound such as can occur after refractive surgery.     

Vitronectin or fibronectin is required for corneal fibroblast-seeded collagen gel contraction

PURPOSE: The wound healing process in the corneal stroma involves the activation of corneal keratocytes and the expression of associated phenotypes (fibroblasts and myofibroblasts). One of these phenotypes, the myofibroblasts, synthesizes alpha-smooth muscle actin in order to affect wound closure by contracting the surrounding matrix. Excessive contraction results in the formation of unresolvable scars that are undesirable in the corneal stroma. The authors tested the effect of vitronectin and fibronectin on the contraction process associated with corneal wound healing. METHODS: Collagen gels were prepared and were exposed to different treatments of fetal calf serum (FCS). The FCS used was either depleted of fibronectin and vitronectin or contained a known concentration of fibronectin, vitronectin, or both at 50 microg/ml. Contraction was measured using image analysis and cross sections of contracted gels were examined for alpha-smooth muscle actin expression using laser confocal microscopy. RESULTS: Fibroblasts seeded in collagen gels paralleled the morphologic characteristics and cell distribution of keratocytes in unwounded cornea. Matrix contraction was dependent on the presence of fibronectin and/or vitronectin where myofibroblasts were present. The cell-mediated contraction process was maximal at 0.5 x 10(5) fibroblasts/ml. CONCLUSIONS: These studies showed that vitronectin or fibronectin is required for the myofibroblast-associated contraction to occur in this in vitro model of stromal wound healing. This model system shows a distinct potential for further studies relating to the corneal wound healing process.     

Microarray studies reveal macrophage-like function of stromal keratocytes in the cornea

PURPOSE: To elucidate biological processes underlying the keratocyte, fibroblast, and myofibroblast phenotypes of corneal stromal cells, the gene expression patterns of these primary cultures from mouse cornea were compared with those of the adult and 10-day postnatal mouse cornea. METHODS: Murine Genome_U74Av2 arrays (Affymetrix Inc., Santa Clara, CA) were used to elucidate gene expression patterns of adult and postnatal day-10 corneal stroma, keratocytes, fibroblasts, and myofibroblasts. RESULTS: Mobilization of stromal cells by culturing led to a wound-healing cascade in which specific extracellular matrix and cornea-transparency-related genes were turned off, and a repertoire of macrophage genes were switched on. Thus, novel transparency-related crystallins detected in the corneal gene expression patterns were downregulated in culture, whereas macrophage genes, mannose receptor type-1, Cd68, serum amyloid-A3, chemokine ligands (Ccl2, Ccl7, Ccl9), lipocalin-2, and matrix metalloproteinase-3 and -12 of innate immunity were induced in primary keratocyte cultures. Fibroblasts expressed the growth-related genes lymphocyte antigen 6 complex locus-A and preprokephalin-1, and myofibroblasts expressed annexin-A8, WNT1-inducible signaling pathway protein-1, arginosuccinate synthetase-1, and procollagen XI of late-stage wound healing. CONCLUSIONS: The emergent biological process suggests a dual role for resident stromal keratocytes in the avascular cornea: one of cornea maintenance, which involves synthesis of proteins related to the extracellular matrix and corneal transparency, and a second of barrier protection macrophage functions, which are switched on during corneal infection and injury.     

The cytokine regulation of SPARC production by rabbit corneal epithelial cells and fibroblasts in vitro

OBJECTIVE: SPARC (osteonectin/BM40) is detected in the corneal stroma during the wound-healing process. To understand the metabolism of SPARC in the cornea, we investigated the effects of cytokines and growth factors on SPARC synthesis by rabbit corneal epithelial cells and fibroblasts. METHODS: Rabbit corneal epithelial cells or fibroblasts were cultured for 3 days with serum-containing minimal essential medium (MEM), then subcultured for 3 days on serum-free MEM with epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta), or interleukin-1beta (IL-1beta). SPARC concentration in the medium was measured by the ELISA method using anti-SPARC monoclonal antibody. RESULTS: The concentration of SPARC in the conditioned medium of the epithelial cells depended on either cell numbers or cultivation periods. When EGF was added to the medium, the amount of SPARC in the medium decreased. The addition of IL-1beta, PDGF, or TGF-beta did not affect SPARC synthesis by the epithelial cells. The production of SPARC by rabbit corneal fibroblasts was low compared with that by epithelial cells. However, the synthesis of SPARC by corneal fibroblasts was significantly enhanced by the addition of TGF-beta. The addition of IL-1beta, PDGF, or EGF slightly increased SPARC synthesis by corneal fibroblasts. CONCLUSIONS: Cytokines and growth factors modulate SPARC synthesis by rabbit corneal epithelial cells and fibroblasts. These results suggest that cytokines and growth factors modulate cell-matrix interaction in corneal wound healing, possibly by regulating SPARC synthesis.     

Increased apoptosis and abnormal wound-healing responses in the heterozygous Pax6+/- mouse cornea

PURPOSE: Corneal wound healing involves a cascade of interactions between the epithelium and stroma. Pax6 is upregulated, and early events include epithelial cell migration and apoptosis of superficial keratocytes. The mouse heterozygous Pax6 (Pax6+/-) corneal phenotype mimics human aniridia-related keratopathy (ARK), and some aspects of wound healing have been shown to be abnormal, including matrix metalloproteinase (MMP)-9 expression. The purpose of this study was to test whether the Pax6+/- genotype affects corneal wound-healing responses, including stromal cell apoptosis, epithelial cell migration rate, and MMP secretion in culture. METHOD: Pax6+/- and wild-type (Pax6+/+) mice were killed and their corneas wounded by epithelial debridement. Whole eyes were cultured in organ culture and corneal epithelial healing rates and keratocyte apoptosis were quantified by topical fluorescein staining and TUNEL, respectively. Dissociated corneal epithelial cells from Pax6+/- and wild-type mice were cultured, and the activities of secreted MMP-9 were determined by zymography. RESULTS: Wound-healing rates during the first 6 hours were significantly faster for larger wounds and for Pax6+/- corneas. Compared with wild-type, wounded Pax6+/- eyes showed significantly more stromal cell apoptosis, and cultured Pax6+/- corneal epithelial cells produced lower MMP-9 activity. CONCLUSIONS: The cumulative effect of abnormal wound-healing responses, characterized by increased stromal cell apoptosis and reduced levels of MMP-9 secretion may contribute to the corneal changes in the Pax6+/- mice. Possible contributions of elevated stromal cell apoptosis and other abnormal wound-healing responses to ARK are discussed.     

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.