Vitronectin supports migratory responses of corneal epithelial cells to substrate bound IGF-I and HGF, and facilitates serum-free cultivation

Vitronectin (VN) is a multi-functional glycoprotein best known for its effects on cell attachment and spreading, but has more recently been shown to mediate cellular responses to growth factors. The presence of VN within the tear film and expression of required receptors (alpha v integrins) on corneal epithelial cells suggests the potential for a similar role within the ocular surface. Thus we have studied the ability of VN to alter the metabolic (MTT assay) and migratory (trans-membrane migration) responses of corneal epithelial cells to growth factors associated with the ocular surface including epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and insulin-like growth factor-I (IGF-I). Our hypothesis was that culture surfaces coated with VN might selectively facilitate responses to growth factors which are known to bind VN including EGF, IGF-I (via IGF binding protein) and HGF. Metabolic responses were observed towards each growth factor when applied to the culture medium, but not towards culture plastic pre-treated with VN and, or growth factors. Optimal metabolic responses were observed towards IGF-I applied in conjunction with EGF. Migration through porous polycarbonate membrane was significantly increased when the substrate had been pre-coated with VN and IGF-I (applied in conjunction with IGFBP-3) or VN and HGF. This finding is consistent with the ability of IGF-I (via an IGFBP) and HGF to form complexes with VN and suggests that integrin/growth factor receptor co-activation is required for corneal epithelial cell migration. In further studies, VN applied in conjunction with IGF-I, IGFBP-3 and EGF (both to the culture plastic and in the culture medium) was found to support the establishment and serial propagation of limbal-corneal epithelial cell cultures in the absence of serum, but irradiated 3T3 cells (i3T3) were still necessary for culture expansion. Immunocytochemistry of resulting cultures for keratin 3 and p63 revealed a similar phenotype to those established under current best-practice conditions (i3T3, foetal bovine serum, EGF and insulin). In conclusion, our novel findings suggest a role for VN-growth factor complexes in stimulating corneal epithelial migration within the provisional wound bed and demonstrate that VN-growth factors interactions can be exploited to enable manufacture of bioengineered ocular surface tissue under serum-free conditions.     

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.     

Correlation of proliferative and anti-apoptotic effects of HGF, insulin, IGF-1, IGF-2, and EGF in SV40-transformed human corneal epithelial cells

The effects of various growth factors on the proliferation and apoptosis of human corneal epithelial cells were investigated. Simian virus 40-transformed human corneal epithelial cells were thus incubated separately with eight different growth factors, after which cell proliferation was evaluated by measurement of [(3)H]thymidine incorporation or with the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay and apoptosis was quantified by the terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick-end labeling assay. Phosphorylation of the protein kinase Akt, which plays an important role in anti-apoptotic signaling, was also assessed by immunoblot analysis. The growth factors examined could be classified into three groups on the basis of their effects on the proliferation and apoptosis of human corneal epithelial cells: hepatocyte growth factor (HGF), insulin, insulin-like growth factor (IGF)-1, IGF-2, and epidermal growth factor (EGF) each increased cell proliferation, inhibited the induction of apoptosis by sodium nitroprusside, and elicited the activation of Akt; transforming growth factor-beta1 and -beta2 inhibited [3H]thymidine incorporation but had no effect on sodium nitroprusside-induced apoptosis or on Akt activity; and platelet-derived growth factor-BB had no effects on the measured parameters. HGF, insulin, IGF-1, IGF-2, and EGF may thus contribute to maintenance of the corneal epithelium and coordinate the proliferative and apoptotic responses of this tissue.     

Transforming growth factor-beta modulates effects of epidermal growth factor on corneal epithelial cells.

In order to understand the mechanisms that bring about maintenance and restoration of the integrity of corneal epithelium, we investigated independent and combined effects of transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF) on rabbit corneal epithelial cells in cell and organ culture. Specifically, we determined whether incubation with these factors influenced 1) cellular proliferation, 2) ability of cells to attach to a fibronectin matrix, and 3) the rate of epithelial migration over corneal stroma. Incubation with TGF-beta caused a dose-related decrease in the incorporation of 3H-thymidine by the epithelial cells. EGF increased 3H-thymidine incorporation, but this effect was antagonized by the addition of TGF-beta into the incubation medium. Incubation with EGF increased the numbers of cells that attached to a fibronectin matrix. TGF-beta itself did not affect the number of attached cells but, again, it antagonized the stimulatory effect of EGF. Similarly, when corneal blocks were cultured with EGF, epithelial migration increased in a dose-related manner. TGF-beta itself did not affect epithelial migration at any of the concentrations tested (0.1-10 ng/ml), but it antagonized EGF-stimulated epithelial migration. These findings suggest that the proliferation and the migration of corneal epithelial cells are regulated by different mechanisms, and that TGF-beta serves as a modulator of the effects of EGF.     

Growth factors: importance in wound healing and maintenance of transparency of the cornea

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.     

Differential expression analysis by gene array of cell cycle modulators in human corneal epithelial cells stimulated with epidermal growth factor (EGF), hepatocyte growth factor (HGF), or keratinocyte growth factor (KGF)

PURPOSE: To identify and differentiate cell cycle and differentiation genes that are up-regulated or down-regulated in human corneal epithelial cells in response to alternative epithelium-modulating cytokines epidermal growth factor (EGF), hepatocyte growth factor (HGF) or keratinocyte growth factor (KGF). METHODS: Primary cultures human corneal epithelial cell (HCE) were treated with 25 ng/ml of EGF, 25 ng/ml HGF, 25 ng/ml KGF, or vehicle for 8 hours. Complementary DNA (cDNA) probes were synthesized from total cellular RNA isolated from the HCE cells. The cDNA probes were hybridized to the Atlas human cell cycle/differentiation array membrane. RNAse protection assay was used to confirm up-regulation of the serine/threonine-protein kinase PITALRE gene by EGF, KGF, and HGF. RESULTS: The expression of one hundred and eleven cell cycle and differentiation genes was monitored with the gene array system. It was found that these epithelial cell-modulating cytokines shared similar effects on some of the cell cycle and differentiation genes that were monitored, but had specific effects on some cytokines. Up-regulation of PITALRE gene expression was confirmed using RNAse protection assay. CONCLUSION: EGF, HGF and KGF had differential effects on cell cycle- and differentiation-related gene expression in corneal epithelial cells. For example, all three mitogenic growth factors up-regulated the expression of cyclin D1 (BCL-1 oncogene) and serine/threonine-protein kinase PITALRE in the primary cultured human corneal epithelial cells. However, EGF and KGF, but not HGF, up-regulated expression of the E2F-1 pRB-binding protein gene. Thus, while these three epithelial mitogens have similar effects on many genes that were analyzed, important differences were noted that may relate to differing effects of these growth factors on corneal epithelial cells. Studies to analyze the significance of the identified differences among these growth factors are in progress.     

The insulin-like growth factor system modulates retinal pigment epithelial cell tractional force generation

PURPOSE: The goal of this study was to determine the influence, if any, of the insulin-like growth factors (IGFs) on retinal pigment epithelial (RPE) cell tractional force generation and the contributions of vitreous insulin-like growth factor-binding proteins (IGFBPs) toward control of growth factor activity. METHODS: IGF effects on RPE were evaluated in tissue culture assays that involved incubation on three-dimensional collagen matrices with responses measured as progressive reduction in matrix thickness. IGFBP effects were evaluated by using the same system, exposing cells to a non-IGFBP-binding growth factor analogue (R(3)IGF-I) or IGFBPs alone or in combination with native growth factors. RESULTS: RPE cells generated tractional forces in response to IGF-I and -II with IGF-I being the more potent stimulus. Differential RPE responses to R(3)IGF-I reflected minor amounts of endogenous IGFBP production. IGFBP-2, -3, and -5 were effective inhibitors of both ligands, whereas IGFBP-6 reduced cell responses to IGF-II only. IGFBP-direct effects on the cells were binding-protein-specific, in that IGFBP-1 had detectable stimulatory effects, and IGFBP-3, -4, -5, and -6 inhibited RPE responses. CONCLUSIONS: IGF-I and -II are potent promoters of RPE cell tractional force generation in vitro. The effects of the six high-affinity IGFBPs on RPE responses are generally inhibitory and protein-specific. IGF ligands and binding proteins are known to be present in the vitreous, the environment that drives RPE responses in proliferative vitreoretinopathy (PVR), suggesting that the IGF system plays a potentially important role in the pathophysiology of this fibrocontractive disease.     

Growth factor mRNA and protein in preserved human amniotic membrane

PURPOSE: To investigate the expression of growth factor mRNA and the level of growth factor protein in preserved human amniotic membrane (AM). METHODS: RT-PCR was used to examine the expression of mRNA for eight growth factors (EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2, -beta3) and two growth factor receptors (KGFR and HGFR) in human AM preserved at -80 degrees C for one month. In addition, ELISAs were used to measure the protein concentrations of seven growth factors (EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2) in preserved human corneas and in AM both with and without amniotic epithelium. RESULTS: RT-PCR revealed that human AM expresses mRNA for EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2, -beta3, KGFR and HGFR, while ELISAs showed that it contains EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2. AM without amniotic epithelium also contains all seven growth factors examined, however, in this tissue the protein levels of EGF, KGF, HGF and bFGF were found to be significantly lower than in native AM. CONCLUSIONS: Preserved human AM expresses mRNAs for a number of growth factors and contains several growth factor proteins that might benefit epithelialization after AM transplantation. High levels of EGF, KGF, HGF and bFGF in AM with amniotic epithelium as compared to AM without amniotic epithelium suggest an epithelial origin for these growth factors. We feel that EGF, KGF and HGF in particular might play important roles in ocular surface wound healing after AM transplantation.     

Characteristics of the human ocular surface epithelium

An appreciation of the biological characteristics of the human ocular surface epithelium affords us a great insight into the physiology of the human ocular surface in health and disease. Here, we review five important aspects of the human ocular surface epithelium. First, we recognize the discovery of corneal epithelial stem cells, and note how the palisades of Vogt have been suggested as a clinical marker of their presence. Second, we introduce the concept of the gene expression profile of the ocular surface epithelium as arrived at using a new strategy for the systematic analysis of active genes. We also provide a summary of several genes abundantly or uniquely expressed in the human corneal epithelium, namely clusterin, keratin 3, keratin 12, aldehyde dehydrogenase 3 (ALDH3), troponin-I fast-twitch isoform, ssig-h3, cathepsin L2 (cathepsin V), uroplakin Ib, and Ca(2+)-activated chloride channel. Genes related to limbal and conjunctival epithelia are also described. Third, we touch upon the genetic abnormalities thought to be involved with epithelial dysfunction in Meesmann's dystrophy, gelatinous drop-like corneal dystrophy, and the ssig-h3-mutated corneal dystrophies. Fourth, we provide an update regarding the current state of knowledge of the role of cytokines, growth factors and apoptosis in relation to ocular surface homeostasis and tissue reconstruction; the main factors being epidermal growth factor (EGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), transforming growth factor-ss (TGF-ss), and some inflammatory cytokines. Fifth, corneal epithelial barrier function and dysfunction as measured by fluorophotometry is remarked upon, with an explanation of the FL-500 fluorophotometer and its ability to detect corneal epithelial dysfunction at a subclinical level. The research described in this review has undoubtedly generated a complete understanding of corneal epithelial pathophysiology-an understanding that, directly or indirectly, has helped advance the development of new therapeutic modalities for ocular surface reconstruction.     

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

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

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.     

Differential effects of epidermal growth factor and interleukin 6 on corneal epithelial cells and vascular endothelial cells.

PURPOSE: Epidermal growth factor (EGF) and interleukin 6 (IL-6) stimulate corneal epithelial wound healing. When applied to the cornea, these cytokines act on various types of cells and therefore may induce corneal neovascularization. We investigated the effects of EGF and IL-6 on cell proliferation and cell migration in rabbit corneal epithelial cells and human umbilical vein endothelial cells (HUVECs). METHODS: Corneal epithelial cells or HUVECs were cultured with EGF or IL-6 in the presence of 1% fetal bovine serum, and the number of cells were counted, or the radioactivity of [3H]thymidine-incorporated cells was measured. Monolayered cultured corneal epithelial cells or HUVECs were mechanically wounded, and then the cells were cultured with serum-free basal medium containing EGF or IL-6. After 12 or 24 h, the wounded area was measured. Corneal blocks were cultured with serum-free TC-199 medium containing EGF or IL-6 for 24 h, and then the length of the path of the corneal epithelium was measured. RESULTS: Estimated cell count and [3H]thymidine uptake showed that EGF stimulated cell proliferation in both corneal epithelial cells and HUVECs in a dose-dependent manner. In contrast, IL-6 did not affect cell proliferation in either cell type. Furthermore, EGF also stimulated cell migration by increasing the monolayer and organ-culture system in both cells in a dose-dependent fashion. However, IL-6 stimulated cell migration only in corneal epithelial cells and not in HUVECs. CONCLUSION: These results demonstrated that EGF stimulated cell proliferation and migration in both corneal epithelial cells and HUVECs. In contrast, IL-6 stimulated only corneal epithelial cell migration and did not affect cell proliferation in either cell type or cell migration in HUVECs. These results suggest that, when applied to the cornea, EGF might induce corneal neovascularization, and IL-6 probably would not.     

表皮生长因子及其受体在眼表创伤愈合中的作用

表皮生长因子及其受体家族是细胞生长分化的重要调节因素。目前已证实表皮生长因子家族成员可持续表达于泪液中,其受体可表达于眼表上皮,对维持眼表的完整和促进角膜组织的创伤愈合具有重要意义。本文就表皮生长因子及其受体家族在眼表创伤愈合中的作用进行综述。     

Growth factor-induced proliferation in corneal epithelial cells is mediated by 12(S)-HETE

PURPOSE: Previous studies in our laboratory have shown that 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), a product of 12-lipoxygenase (12-LOX) activity, is the predominant metabolite formed in rabbit corneas after injury. The present study was undertaken to investigate the effects of epidermal growth factor (EGF), hepatocyte growth factor (HGF), and keratinocyte growth factor (KGF) on 12-LOX expression and activity. We also investigated whether 12(S)-HETE mediated the growth factor-induced proliferation of corneal epithelial cells. METHODS: Rabbit corneas were stimulated with EGF, HGF, and KGF (10 ng ml(-1)) for different times. 12-LOX activity was assayed by incubating corneal microsomal preparations with radiolabeled arachidonic acid (AA) as substrate. For inhibitor studies, the microsomes were pretreated with 12-LOX-specific inhibitors baicalein (BC) or cinnamyl 3,4-dihydroxy-(alpha)-cyanocinnamate (CDC). Lipid extracts were injected onto an Ultramex 5 microm C(18) column and radioactivity was monitored online by a Radiomatic Flo-One Beta detector. Stereochemical analysis of 12-HETE product was determined by chiral-phase HPLC. To evaluate the effects of growth factors on 12-LOX mRNA expression, mRNA was extracted at several time points (12, 24, 36, 48 hr) and subjected to real-time PCR. For 12-LOX protein expression, microsomal preparations from 24- and 48-hr incubations were analyzed by Western blot. In cell-proliferation studies, epithelial cells treated with EGF, HGF, or KGF for 24, 48, and 72 hr were measured with a CyQUANT cell-proliferation assay kit. To determine the role of growth factor-induced 12(S)-HETE synthesis on corneal epithelial cell proliferation, cells were pretreated with 12-LOX-specific inhibitors BC or CDC prior to growth-factor supplementation. RESULTS: Stimulation with EGF, HGF, or KGF for 12 hr induced 12-LOX mRNA expression in rabbit corneal epithelial cells. This gene induction was followed by an increase in protein expression at 24 and 48 hr and a marked increase in 12(S)-HETE synthesis when compared to untreated controls. At 24-hr incubations, KGF showed a greater capacity than did EGF and HGF to stimulate microsomal 12-LOX activity, while at 48 hr 12(S)-HETE synthesis was significantly greater in EGF-treated cells as compared to that of HGF- and KGF-treated cells. Pretreatment with 12-LOX inhibitors blocked the growth factor-induced increase in 12(S)-HETE synthesis. Stimulation with growth factors or 12(S)-HETE for 24, 48, and 72hr produced a significant increase in corneal epithelial proliferation, which was partially inhibited by pretreatment of cells with 12-LOX-specific inhibitors. CONCLUSION: These findings suggest that EGF, HGF, and KGF stimulate 12(S)-HETE production in rabbit corneal epithelial cells through gene induction of 12-LOX. Furthermore, 12(S)-HETE may play a role in regulating epithelial cell proliferation and the rate of corneal re-epithelialization following an injury.     

Combined effect of extracellular matrices and growth factors on bovine corneal endothelial cells in culture

PURPOSE: First, to confirm that corneal endothelial cells in the confluent state have the capability to form cellular covering. Second, to establish a method to study the combined effect of extracellular matrices (ECMs) and growth factors on the biological response in corneal endothelial cells in culture. METHODS: Bovine corneal endothelial cells were cultured inside a cylinder set on a plastic dish. They formed a confluent cell nest on the dish coated with type I or type IV collagen, laminin, or fibronectin. After the removal of the cylinder, hepatocyte growth factor (HGF), epidermal growth factor, transforming growth factor-alpha or transforming growth factor-beta(1) was added to the cultures. Each confluent cell nest enlarged outward, and its increased area size was measured. Cellular response in the nest, including cellular proliferation, was analyzed. RESULTS: The size of the increased area of the culture on type IV collagen plus HGF was the largest of all the combinations of ECMs and growth factors. The responses of component cells in the increased area consisted of cellular hypertrophy, proliferation, migration and giant cell formation. The treatment with type IV collagen plus HGF clearly promoted all the above responses. CONCLUSIONS: The biological response of corneal endothelial cells was regulated by ECMs and growth factors.     

Protein kinase C alpha and epsilon differentially modulate hepatocyte growth factor-induced epithelial proliferation and migration

Protein kinase C (PKC) isoenzymes require membrane translocation for physiological activation. We have recently shown that the growth factors such as epidermal growth factor and hepatocyte growth factor (HGF), but not keratinocyte growth factor (KGF), regulate PKCalpha activation to promote epithelial wound healing [Sharma, G.D., Ottino, P., Bazan, H.E.P., 2005. Epidermal and hepatocyte growth factors, but not keratinocyte growth factor, modulate protein kinase C alpha translocation to the plasma membrane through 15(S)-hydroxyeicosatetraenoic acid synthesis. J. Biol. Chem. 280, 7917--924]. Protein kinase C alpha (PKCalpha) and protein kinase C epsilon (PKCvarepsilon) are two differentially regulated isoenzymes. While PKCalpha requires Ca(2+) for its activation, PKEvarepsilon is Ca(2+) independent. However, growth factor-induced activation of these enzymes and their specific regulation of epithelial migration and proliferation have not been explored. In the present study, we overexpressed PKCvarepsilon fused to green fluorescent protein to examine its translocation in real-time to the plasma membrane in living human corneal epithelial cells. Stimulation with HGF and KGF demonstrated translocation of PKCvarepsilon to the plasma membrane. Because HGF activates both PKCs, this growth factor was used to stimulate wound healing. PKCalpha or PKCvarepsilon-genes were knocked down individually without affecting the basal expression of the other PKC isoforms. Gene knockdown of PKCalpha significantly inhibited HGF-stimulated proliferation of human corneal epithelial cells. In contrast, PKCvarepsilon-gene-silencing severely impaired the HGF-stimulated migratory ability of human corneal epithelial cells. When migrating epithelial cells in the cornea wound bed after injury were transfected with specific PKCalpha- or PKCvarepsilon-siRNA, there was a significant delay in wound healing. Corneal wound healing stimulated with HGF in similar conditions was also inhibited. On the other hand, overexpression of PKCalpha or PKCvarepsilon-genes fused with green fluorescent protein in migrating corneal epithelium accelerated repair of the epithelial defect. Our findings demonstrate that PKCalpha and PKCvarepsilon modulate different stages of wound healing stimulated by HGF and contribute to epithelial repair by playing selective regulatory roles in epithelial proliferation and migration, both crucial to corneal wound healing.     

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

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

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.     

In vitro effects of three blood derivatives on human corneal epithelial cells

Purpose. We compared the effects of three blood derivatives, autologous serum (AS), platelet-rich plasma (PRP), and serum derived from plasma rich in growth factors (PRGF), on a human corneal epithelial (HCE) cell line to evaluate their potential as an effective treatment for corneal epithelial disorders. Methods. The concentrations of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and fibronectin were quantified by ELISA. The proliferation and viability of HCE cells were measured by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. Cell morphology was assessed by phase-contrast microscopy. The patterns of expression of several connexin, involucrin, and integrin α6 genes were analyzed by real-time RT-PCR. Results. We found significantly higher levels of EGF in PRGF compared to AS and PRP. However, AS and PRGF induced robust proliferation of HCE cells. In addition, PRGF cultured cells grew as heterogeneous colonies, exhibiting differentiated and non-differentiated cell phenotypes, whereas AS- and PRP-treated cultures exhibited quite homogeneous colonies. Finally, PRGF upregulated the expression of several genes associated with communication and cell differentiation, in comparison to AS or PRP. Conclusions. PRGF promotes biological processes required for corneal epithelialization, such as proliferation and differentiation. Since PRGF effects are similar to those associated with routinely used blood derivatives, the present findings warrant further research on PRGF as a novel alternative treatment for ocular surface diseases.