Requirement of microtubule assembly for initiation of EGF-stimulated corneal epithelial migration

To understand the mechanisms of epidermal growth factor (EGF)-stimulated re-epithelialization, the roles of microtubules and microfilaments in epithelial migration were investigated using the organ culture of rabbit cornea. The action of EGF was also compared with that of fibronectin. The addition of either EGF or fibronectin increased the epithelial sheet migration. Nocodazole, an inhibitor of the microtubule assembly, alone did not affect the epithelial migration. Nocodazole antagonized the EGF-dependent increase of the epithelial migration when nocodazole coexisted during the period of delivery of the signal from EGF. However, the stimulatory action of fibronectin was observed even in the presence of nocodazole. On the other hand, cytochalasin B, inhibitor of the microfilament assembly, inhibited the epithelial migration in a dose-dependent manner regardless of whether EGF or fibronectin was present or not. These results indicated that the formation of microfilaments was essential for the corneal epithelial migration and that the stimulatory action of EGF on the corneal epithelial migration depended on the assembly of microtubules. Therefore, in corneal epithelial wound healing, the action of EGF requires the organization of both microfilaments and microtubules, while that of fibronectin requires the reorganization of the microfilaments.     

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.     

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.     

Hyaluronan stimulates corneal epithelial migration.

Hyaluronan (hyaluronic acid), well-known for its viscoelastic properties, is also recognized as a biological signal to cells. Using organ cultures of the rabbit cornea, we investigated the effects of hyaluronan on the migration of corneal epithelium. The addition of hyaluronan to the culture medium increased the length of the path of the corneal epithelial layer in a dose-dependent fashion. Other glycosaminoglycans (chondroitin, chondroitin sulphate, keratan sulphate and heparan sulphate) were also tried, but only hyaluronan exhibited a stimulatory effect on corneal epithelial migration. The effects of hyaluronan and fibronectin or epidermal growth factor (EGF) were additive; the addition of antisera against fibronectin or against EGF did not alter the stimulatory effect of hyaluronan. These results demonstrate that hyaluronan stimulates corneal epithelial migration by mechanism(s) different from those of fibronectin and EGF.     

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.     

Fibronectin synthesis by the rabbit cornea: effects of mouse epidermal growth factor and cyclic AMP analogs

We investigated whether the cornea can synthesize fibronectin which participates in corneal wound-healing. We also examined the effects of epidermal growth factor (EGF) and cyclic AMP analogs on fibronectin synthesis by the cornea. Rabbit corneal blocks were cultured in medium without serum, and the amount of fibronectin in culture medium was determined by ELISA assay. When rabbit corneal blocks were cultured in the medium alone, fibronectin contents in the medium increased with increase in the incubation period. This increase was enhanced by the addition of EGF to the culture medium, and was inhibited by addition of a protein synthesis inhibitor, cycloheximide. The addition of cAMP analogs, 8-bromo cyclic-AMP and dibutyryl cAMP, to the culture medium also increased the rate of fibronectin production by the cornea. These results show that the cornea can synthesize fibronectin and its synthesis is stimulated by EGF and cAMP analogs.     

Effects of atrial natriuretic peptide and sodium nitroprusside on epidermal growth factor-stimulated wound repair in rabbit corneal epithelial cells

PURPOSE: Treatment of rabbit corneal epithelial cells (RCEC) with epidermal growth factor (EGF) stimulates cell proliferation and wound repair in a cell culture model system. Studies have also shown that atrial natriuretic peptide (ANP) and sodium nitroprusside (SNP), a nitric oxide-generating agent, inhibit proliferation of a variety of cell types. The aim of the present work was to examine whether ANP or SNP has any effect on EGF-stimulated proliferation of RCEC involved in wound repair. METHODS: The SV-40 immortalized RCEC were cultured in 24-well plates until they became confluent. Wounds of uniform size (8 mm diameter) were created and the cells allowed to grow in the presence and absence of EGF and/or other agents. At prescribed time intervals, the cells were stained by Giemsa and the wound areas digitized and quantified by Sigma Image Scan System. The cGMP contents in RCEC, treated with or without ANP or SNP, were measured by radioimmunoassay. RESULTS: Addition of EGF (1-100 ng/ml) to RCEC stimulated cell proliferation which significantly reduced the time required for wound closure. Addition of ANP (1 nM to 10 microM) or SNP (10 microM to 1 mM), in the presence of EGF, dose-dependently inhibited the growth factor-stimulated wound closure in RCEC. When added alone to the cells, ANP or SNP increased cGMP accumulation in a dose-dependent manner. Addition of ANP (1 microM) or SNP (1 mM) to primary corneal epithelial cells, in the presence and absence of EGF, also inhibited the wound closure with a corresponding increase in cGMP contents. Treatment of the cells with ODQ (10 nM to 10 microM), a soluble guanylate cyclase inhibitor, dose-dependently decreased the SNP-induced accumulation of cGMP, and reversed the inhibitory effect of SNP on EGF-stimulated wound closure. Addition of membrane-permeable cGMP analog, 8-bromo-cGMP, to RCEC inhibited the EGF-stimulated wound closure in a dose-dependent manner. Treatment of RCEC with mitomycin C (5 microM) exerted a marked inhibitory effect on wound closure in the presence and absence of EGF, and also abrogated the inhibitory effect of 8-bromo-cGMP on wound closure in the EGF-treated and untreated cells. CONCLUSIONS: The results demonstrate that ANP and SNP inhibit the EGF-stimulated wound repair in RCEC. The effect of these agents is mediated via activation of guanylate cyclases that generate cGMP. Cyclic GMP appears to exert its inhibitory effect at the level of cell proliferation and not cell migration. The data suggest an important role for cGMP-dependent protein kinase in proliferation of RCEC stimulated by EGF.     

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

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

Chemotactic and haptotactic activities of fibronectin for cultured rabbit corneal epithelial cells

A previous study reported that both fibronectin and epidermal growth factor (EGF) stimulated corneal epithelial resurfacing. Fibronectin appears in the cornea after injury, and corneal epithelial cells migrate over the temporary fibronectin matrix. To determine whether fibronectin serves as chemoattractant and haptoattractant for the directed movement of corneal epithelial cells, the directed migration of cultured rabbit corneal epithelial cells was measured in vitro using a Boyden chamber. Chemotactic and haptotactic migration were assayed separately. Fibronectin was found to stimulate attachment of corneal epithelial cells and to have chemotactic, haptotactic and chemokinetic activities for the corneal epithelial cells. In contrast, EGF had no chemotactic activity.     

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

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

细胞因子对角膜成纤维细胞生长的作用

目的,研究细胞团子对角膜成纤维细胞的生长作用,为细胞因子治疗角膜疾病的机制研究提供实验依据。方法：用MTF法测定细胞因子EGF、GTFF－β_1、FN等对角膜成纤维细胞生长的影响作用,结果：EGF对角膜成纤维细胞具有生长调节作用,在0.01~1.0μg/ml浓度下,具有明显促生长作用（与0浓度组比较P＜0．001）,其最佳浓度为0．1μg/ml,但浓度在10.0μg／ml时却对其具有明显抑制作用（与0浓度组比较P＜0005）,TGF－β_1（0.01－10.0ng/ml）、FM（0.01－10．0mg/ml）对角膜成纤维细胞未见明显促生长或抑制作用（P＞0.05）结论：EGF能调节角膜成纤维细胞生长,而TGF、FN对角膜成纤维细胞生长无影响,     

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.     

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

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

Effect of growth factors with dexamethasone on healing of rabbit corneal stromal incisions

Treatment of rabbit corneal wounds with topical corticosteroid retards both epithelial regeneration and healing of penetrating stromal wounds. Currently, no clinical agent is available which accelerates the rate of stromal wound healing. Epidermal growth factor (EGF, 0.5 mg ml-1), fibroblast growth factor (FGF, 20 micrograms ml-1), and insulin (0.5 mg ml-1) were tested for their ability to accelerate healing of totally penetrating wounds in rabbit corneas when the hormones were administered alone or in combination with dexamethasone (1 mg ml-1). After 5 days of treatment with eye drops, the tensile strengths of corneal wounds treated with EGF (54 +/- 4 g mm-1) or treated with EGF and dexamethasone (32 +/- 9 g mm-1) were significantly higher than the tensile strengths of corneal wounds treated with only saline vehicle (3 +/- 1 g mm-1) or dexamethasone (1 +/ 0 g mm-1) (P less than 0.001). The combination of dexamethasone with EGF significantly (P less than 0.025) reduced the strength of corneal wounds compared to treatment with EGF alone. Similarly, the tensile strength of corneal wounds after 5 days of insulin treatment alone (28 +/- 8 g mm-1) or in combination with dexamethasone (25 +/- 7 g mm-1) was significantly increased compared with saline- or dexamethasone-treated corneas (P less than 0.001). In the absence of dexamethasone, EGF increased the tensile strength of corneal wounds significantly better than insulin (P less than 0.01). However, when EGF or insulin were given in combination with dexamethasone there was no significant difference between the tensile strength produced by the peptide hormones. In comparison to the tensile strength of corneal wounds treated by EGF or insulin, treatment with FGF alone (5 +/- 4 g mm-1) or in combination with dexamethasone (2 +/- 1 g mm-1) produced poor wound healing. The in vitro actions of EGF or FGF alone or in combination with dexamethasone were tested for ability to stimulate [3H]-thymidine incorporation into pure cultures of human corneal fibroblasts (HCF) in defined culture medium. EGF (5 mM) or FGF (100 ng ml-1) alone stimulated [3H]-thymidine incorporation approximately 2.5-fold compared to control cultures, whereas in combination with dexamethasone (10 nM), the stimulatory action of FGF, but not EGF, was abolished. Dose-response curves indicated that HCF in culture were very sensitive to EGF, insulin, and FGF with maximum stimulation of [3H]-thymidine incorporation occurring at approximately 1 nM for EGF and insulin and at 100 micrograms ml-1 for FGF. Binding of 125I-EGF to HCF reached maximum after 2 hr at 37 degrees C and was specific, saturable, and of high affinity (half saturation at 1 nM). (ABSTRACT TRUNCATED AT 400 WORDS)     

Synergistic effects of hyaluronan and fibronectin on epithelial migration in rabbit cornea in vitro

PURPOSE: We investigated the interactions of hyaluronan with extracellular matrix proteins (fibronectin, laminin, and type IV collagen) on corneal epithelial migration by using an organ-culture system of rabbit corneas. METHODS: Rabbit corneal blocks were cultured in the absence or presence of various reagents for a total of 20 h. The corneal blocks were then fixed, dehydrated, embedded in paraffin, and stained by hematoxylineosin, and the length of the path of epithelial migration was measured. RESULTS: The addition of hyaluronan, fibronectin, or type IV collagen alone stimulated epithelial migration in a dose-dependent manner, but laminin did not. When corneal blocks were pretreated with extracellular matrix proteins (fibronectin, laminin, or type IV collagen) for 5 h and then were cultured for 15 h with hyaluronan, only pretreatment with fibronectin demonstrated a synergistic effect with hyaluronan on corneal epithelial migration. In contrast, when corneal blocks were pretreated with hyaluronan for 5 h and then cultured for 15 h with extracellular matrix proteins, the additive effects of hyaluronan with fibronectin or hyaluronan with type IV collagen were observed. The synergistic effects of hyaluronan and fibronectin on corneal epithelial migration were partially inhibited by the addition of antiserum against fibronectin. In contrast, the addition of antiserum against fibronectin receptor completely inhibited the synergistic effects of hyaluronan and fibronectin on epithelial migration. CONCLUSION: These results suggest that hyaluronan and fibronectin have a synergistic effect, with fibronectin pretreatment augmenting hyaluronan-stimulated corneal epithelial migration.     

EGF联合KGF促进人角膜上皮细胞生长

目的:寻找促进角膜上皮损伤修复,治疗持续性角膜上皮缺损的有效药物方法:用~3H—胸腺嘧啶核苷(~3H—TDR)掺入及液体闪烁技术,观察外源性表皮生长因子(EGF)联合角蛋白细胞生长因子(KGF)对体外培养的角膜上皮细胞DNA合成的影响,并计算细胞倍增时间。结果:10ng/mlEGF,10ng/mlKGF单独或联合应用均有明显促进人角膜上皮细胞DNA合成的作用(与对照组比较 P<0.01),联合用药,作用更强(P<0.05)。应用EGF与KGF明显缩短了细胞倍增时间。结论:外源性EGF与KGF对体外培养的人角膜上皮细胞有明显的促细胞增生作用,联合用药,效果更佳。表明EGF与KGF具有应用于临床,促进角膜上皮损伤修复的可能性。眼科学报1996; 12:107-109。     

EGF促进RPE细胞β2肾上腺能受体诱导的cAMP形成：受体间交互作用分析

研究证实表皮生长因子(EGF)不影响培养人眼视网膜色常上皮(RPE)细胞cAMP的基础水平，但促进异丙肾上腺索激活β₂受体后诱导cAMP水平升高的效应，井呈量效信赖关系。EGF对腺嘌呤核苷A₂受体激动剂NECA诱导cAMP水子升高的效应没有明显影响．细胞增殖分析表明，EGF刺激人眼RPE细胞增殖，而DibutyrylcAMP和异丙肾上腺素抑制RGF刺激增殖的效应。结果提示，在人眼RPE细胞EGF和β₂受体之间存在受体间交互作用． （中华眼底病杂志，1995，11：25-27）