表皮生长因子对人角膜内皮细胞损伤修复的影响

目的　观察人表皮生长因子 (epidermal growth factor,EGF)对人角膜内皮细胞损伤修复的影响 ,检测人角膜内皮细胞表皮生长因子受体 (epidermal growth factor recep-tor,EGFR)的表达。方法　胎儿角膜内皮细胞体外培养传一代融合后 ,定量损伤细胞 ,倒置显微镜下观察不同浓度 EGF对角膜内皮细胞损伤修复的影响 ,并于损伤前及损伤后 1、3、7、14 d用间接免疫荧光的方法检测角膜内皮细胞 EGFR的表达。结果　 EGF促进人角膜内皮细胞损伤愈合 ,并显示剂量依赖性 ,EGF在 10 μg· L- 1时促进作用达高峰 ,浓度再增高促进作用下降。人角膜内皮细胞损伤前及损伤后均在细胞膜上表达 EGFR,表现为细胞膜上绿色荧光 ,在未损伤区的细胞及缺损区修复的细胞均见表达。结论　 EGF促进人角膜内皮细胞损伤修复 ,人角膜内皮细胞表达 EGFR     

表皮生长因子对不同区域角膜上皮细胞体外传代培养的影响

目的　观察表皮生长因子 (EGF)对角膜上皮细胞体外传代培养的影响。方法　用抗 Brdu抗体标记法 ,流式细胞仪检测细胞增殖情况。结果　对照组 :中央部细胞传代次数为 ( 5± 1)代 ,周边部的为 ( 7± 1)代 ,角膜缘部的为 ( 8±2 )代。EGF组 :角膜中央及周边部上皮细胞传代次数分别提高 ( 1± 1)及 ( 2± 1)代 ;角膜缘部的增加 ( 5± 1)代。结论　角膜缘细胞在添加EGF后 ,细胞体外传代有显著提高 (P <0 .0 1)。由此提示 :通过进一步改善培养条件 ,体外延长角膜缘部细胞的存活是可能的     

The coating of bovine and rabbit corneas denuded of their endothelium with bovine corneal endothelial cells.

A method for coating corneas denuded of their endothelium has been developed. The attachment and spreading of cultured bovine corneal endothelial cells seeded upon the Descemet's membrane of corneal buttons previously denuded of their endothelium by delicately sweeping the endothelial side with a cotton swab have been analyzed. Confluent cultures of bovine corneal endothelial cells were exposed to trypsin to disrupt the cell monolayer into single cells. Increasing concentrations of endothelial cells ranging from 2·5 × 10⁴ to 3 × 10⁵ cells were then seeded on the denuded Descemet's membrane of 11 mm bovine corneal buttons. When the corneal buttons were stained with alizarin red after an incubation period of 8 hr at 37°C, the best coating was observed with 10⁵ seeded cells. In this case, no areas of denudation could be seen and the cells were clearly apposed to one another, thereby reconstituting an endothelial cell monolayer. The cultured bovine corneal endothelial cells seeded on denuded Descemet's membranes plated extremely rapidly. By 15 min, 80% of Descemet's membrane was covered with a monolayer of endothelial cells and by 30 min all of Descemet's membrane was covered.The plating of bovine corneal endothelial cells on denuded Descemet's membrane was a direct function of the trypsin concentration to which they were first exposed. Cells first treated with 0·05% trypsin plated poorly 1 hr after being seeded on a denuded Descemet's membrane. Better plating efficiency was achieved with cells first exposed respectively to 0·025% and 0·01% trypsin. The best results were consistently obtained with cells first dissociated with 0·005% trypsin.Although serum is required in vitro for the attachment of normal cells to tissue culture dishes, it was not required for the attachment of corneal endothelial cells to the denuded Descemet's membrane. Cultured corneal endothelial cells plated equally well in the presence or absence of serum. Similar results were obtained when the cells were suspended in aqueous humor instead of in tissue culture medium.When denuded rabbit corneas were used as a substrate instead of bovine corneas, all the parameters studied for the attachment and spreading of bovine corneal endothelial cells seeded on bovine corneas (cell density, time, and medium) lead to similar conclusions with respect to the interactions between corneal endothelial cells and rabbit Descemet's membrane.     

Role of epidermal growth factor receptor (EGFR) in corneal remodelling in diabetes

Purpose: This study examined the role of epidermal growth factor receptor (EGFR) signalling on the organization and remodelling of collagen fibrils (CFs) and proteoglycans (PGs) in the stroma of diabetic rat cornea. Methods: Diabetes was induced in female Wistar rats (n = 5) by streptozotocin (STZ) injection (55 mg/kg). Treatment with a selective inhibitor of EGFR tyrosine kinase, AG1478, was started on the same day as the induction of diabetes and administered every other day for 4 weeks. Corneas were fixed in 4% paraformaldehyde at 4 ° to allow for analysis of CF diameters and in 2.5% glutaraldehyde in sodium acetate buffer containing cuprolinic blue to enable the study of PG distribution. AnalySIS soft imaging software was used to analyse CFs and PGs. Results: Epithelial thickness, and median diameter and area fraction of CF in corneal stroma were decreased in diabetic rat cornea compared with normal cornea (p < 0.001), whereas the median PG area and area fractions were significantly increased (p < 0.001). Treatment with AG1478, although it had no action on normal cornea, prevented these diameter and area fraction changes in CFs and PGs. The cornea of AG1478‐treated diabetic rats showed a slight increase in CF diameter and area fraction and a decreased number density. Conclusions: These data show that the distribution of corneal stroma CFs and PGs was altered after 4 weeks of diabetes and that, furthermore, treatment with an EGFR signalling inhibitor normalized these abnormalities. The data suggest that EGFR plays an important role in the development of diabetes‐induced corneal remodelling.     

Growth factors in the anterior segment: role in tissue maintenance, wound healing and ocular pathology

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

The effect of an Eye Derived Growth Factor (EDGF) on corneal epithelial regeneration

EDGF an Eye Derived Growth Factor, purified from bovine retina is shown to increase significantly the rate of healing of rabbit corneal epithelium wounded by Iodine vapours. Conversely other commercially available healing substances had no effect. EDGF at 1 mg/ml and 10 μg/ml were equally effective in the long term to accelerate the complete epithelium reorganization. These results tend to confirm the values of in vitro studies on the relationship between growth factors and the control of basement membrane synthesis.     

持续性角膜上皮缺损的治疗进展

持续性角膜上皮缺损(PED/PCEDs)是指角膜损伤后10～14d内,在接受了相应治疗后,角膜也未能迅速重新形成上皮并闭合而导致的一种角膜疾病。角膜上皮的破坏和基质层的损伤容易使眼部受到感染、发生基质溃疡、穿孔、瘢痕,甚至丧失视力。就目前而言,临床医生对PED的治疗仍然面临相当大的挑战。标准的治疗方法包括配戴绷带隐形眼镜和使用人工泪液治疗,而新开发的药物则可以通过促进各类生长因子的生成使角膜重新形成上皮,进一步配合相应外科手术为角膜提供神经支配,以此达到治疗的效果。此外,确诊PED后应尽早接受治疗,以避免继发性并发症。本文就PED的流行病学、病因学、诊断与临床表现、治疗方法及预后进行综述。     

人胎儿晶状体上皮细胞增殖及相关因子的动态研究

目的：观察增殖细胞核抗原（PCNA）及转化生长因子－β1（TGF－β1），表皮生长因子（EGF）在人胎和晶状体上皮细胞的表达，并探讨其在晶状体发育中的作用。方法：用SP免疫细胞化学法对48例人胎儿不同胎龄晶状体上皮细胞进行PCNA，TGF－β1，EGF免疫细胞化学染色。结果：PCNA，TGF－β1，EGF在人胎儿不同胎龄状体上皮细胞内均有表达；免疫阳性细胞随着胎龄的增加而减少。PCNA阳性细胞核为桔黄色或棕黄色；TNF－β1，EGF阳性细胞浆为桔共色或棕黄色的颗粒状或斑块状；经相关分析，TGF－β1与EGF以及TGF－β1，EGF与PCNA均呈正相关。结论：PNCA，TGF－β1，EGF在人胎儿不同胎龄晶状体上皮细胞内均有表达，TGF－β1，EGF具有协同作用，共同促进晶状体上皮细胞的增殖。     

(R,R)-XY-10和(S,S)-XY-10诱导的视网膜色素上皮细胞增生作用及其作用机制(英文)

目的:观察(R, R)-XY-10和(S, S)-XY-10对视网膜色素上皮细胞的增生作用,并且进一步的研究其作用机制。但(R, R)-XY-10和(S, S)-XY-10对人脐静脉内皮细胞并无增生的作用。方法:通过人视网膜色素上皮细胞(ARPE-19)和人脐静脉内皮细胞(HUVECs)研究(R, R)-XY-10和(S, S)-XY-10对视网膜色素上皮细胞的增生作用,并且采用ERK、KT、PI3K、蛋白激酶C(PKC)和一氧化氮合酶(NOS)抑制剂来研究其作用机制。结果:(R, R)-XY-10和(S, S)-XY-10促进了ARPE-19细胞的增生,并具有剂量依赖性,但是对HUVECs细胞没有影响。如果同时加入增生抑制剂H7 (5μmol/L)、金丝桃素(20μmol/L)、PD98059(2μmol/L)、LY294002(50μmol/L)、SH-5(10μmol/L)和L-NAME (100μmol/L),则给予H7、金丝桃素、PD98059和LY294002各组的增生作用受到了抑制,而给予SH-5和L-NAME两组的增生作用没有影响。结论:(R, R)-XY-10和(S, S)-XY-10能够诱导ARPE-19细胞增生,其作用可能是通过MAPK和PI3K的途径来发挥该作用。因此,(R, R)-XY-10和(S, S)-XY-10能通过修复损伤的RPE细胞来治疗老年性黄斑变性。     

人晶状体上皮细胞HGF与c-Met的表达对增殖和凋亡的影响

目的：探讨肝细胞生长因子(hepatocyte growth factor,HGF)在人晶状体上皮细胞(lens epithelial cell,LEC)的表达及其对晶状体上皮细胞的促增殖和抑制凋亡的作用。方法：取原代培养人晶状体上皮细胞,应用RT-PCR,Western blot检测HGF,C-Met的mRNA及蛋白的表达,应用MTT法检测HGF对晶状体上皮细胞增殖的影响,Western-blot检测：Bcl-2的蛋白表达,以揭示其对晶状体上皮细胞凋亡的影响。结果：HGF和c-Met在人晶状体上皮细胞有表达,HGF有促进晶状体上皮细胞增殖的作用,并能抑制凋亡产生。结论：HGF参与人晶状体上皮细胞的增殖代谢过程,与晶状体上皮细胞的增殖相关。     

Protection of human corneal epithelial cells from hypoxia-induced disruption of barrier function by hepatocyte growth factor

The barrier function of the corneal epithelium maintains corneal homeostasis and is mediated by tight junctions (TJs) and adherens junctions (AJs). It is also susceptible to disruption by hypoxia. We have now examined the effects of hypoxia on TJs and AJs as well as on barrier function in human corneal epithelial (HCE) cells. Moreover, we investigated whether such effects of hypoxia might be modulated by hepatocyte growth factor (HGF). The subcellular distribution of the TJ proteins ZO-1 and occludin, the AJ proteins E-cadherin and β-catenin, and actin filaments was examined by fluorescence microscopy. The abundance of junctional proteins as well as of myosin light chain kinase (MLCK) was determined by immunoblot analysis. Barrier function was evaluated by measurement of transepithelial electrical resistance (TER). Hypoxia-induced both the disappearance of ZO-1 from the borders of neighboring HCE cells as well as the down-regulation of ZO-1 expression without affecting the distribution or abundance of occludin, E-cadherin, or β-catenin. It also induced the formation of actin stress fibers, the up-regulation of MLCK expression, and a reduction in the TER of HCE cells. All these effects of hypoxia were inhibited by HGF. Neither hypoxia nor HGF exhibited a mitogenic or cytotoxic effect on HCE cells. HGF thus protects HCE cells from hypoxia-induced disruption of barrier function by maintaining the expression and distribution of ZO-1. Inhibition of the effects of hypoxia on the organization of the actin cytoskeleton might also contribute to this protective action of HGF.     

Epithelial-stromal interactions: specific stimulation of corneal epithelial cell growth in vitro by a factor(s) from cultured stromal fibroblasts

By using a recently modified method of isolating and culturing rabbit corneal cells, this study investigated the presence of a diffusible substance(s) in stromal fibroblast conditioned medium that stimulated the growth of cultured corneal epithelial cells. The growth stimulation involved initiation of DNA synthesis (assayed by [3H]-thymidine incorporation) and enhanced cell proliferation (quantified by cell counting). Among the three corneal cell types, only fibroblasts (rabbit and human) released the stimulatory substance, which acted only on epithelial cells. The effect of this stromal fibroblast factor (SFF) was observed after an exposure period of less than 16 hr and persisted as long as it was present. Its action was concentration-dependent and was not a result of improvement in the survival of epithelial cells during culture. Both sparse and confluent epithelial cultures were susceptible to SFF. The release of SFF was correlated with the number of fibroblasts in the culture and appeared to be sensitive to the growth condition of the cells. Both the release and action of SFF did not depend on the presence of serum in the culture medium. The factor was heat resistant and insensitive to proteolytic enzymes. From ultrafiltration studies, the size of SFF was estimated to be in the approximate range of 50-1000 daltons. By direct comparison of the stimulatory effect with other previously studied growth promoting agents, it was concluded that SFF was not epidermal growth factor, fibroblast growth factor, putrescine, cyclic AMP, hydrocortisone or acetate. The implication of SFF in the regulation of epithelial growth by endogenous, intercellular mechanisms is discussed.     

Inhibition of corneal neovascularization with plasmid pigment epithelium-derived factor (p-PEDF) delivered by synthetic amphiphile INTeraction-18 (SAINT-18) vector in an experimental model of rat corneal angiogenesis

The use of Synthetic Amphiphile INTeraction-18 (SAINT-18) carrying plasmid pigment epithelium-derived factor (p-PEDF) as an anti-angiogenesis strategy to treat corneal neovascularization in a rat model was evaluated. Four partially dried forms (Group A: 0 μg, B: 0.1 μg, C: 1 μg, D: 10 μg) of a p-PEDF-SAINT-18 were prepared and implanted into the rat subconjunctival substantia propria 1.5 mm from the limbus at the temporal side. The 1 μg of plasmid-basic fibroblast growth factor--SAINT-18 (p-bFGF-SAINT-18) (1 μg) was prepared and implanted into the rat corneal stroma 1.5 mm from the limbus on the same side. Inhibition of neovascularization was observed and quantified from day 1 to day 60. PEDF (50-kDa) and bFGF (18-kDa) protein expression were analyzed by biomicroscopic examination, Western blot analysis, and immunohistochemistry. Gene expression in corneal and conjunctival tissue was observed as early as 3 days after gene transfer and stably lasted for over 3 months with minimal immune reaction. Subconjunctival injection of a highly efficient p-PEDF-SAINT-18 successfully inhibited corneal neovascularization. Successful gene expression of bFGF, PEDF and a mild immune response of HLA-DR were shown by immunohistochemistry staining. We concluded that SAINT-18 was capable of directly delivering genes to the ocular surface by way of subconjunctival injection, and delivered sustained, high levels of gene expression in vivo to inhibit angiogenesis.     

Functional expression of transient receptor potential vanilloid 3 (TRPV3) in corneal epithelial cells: Involvement in thermosensation and wound healing

Transient receptor potential vanilloid 3 (TRPV3), a member of the calcium-permeable thermosensitive TRP (thermoTRP) subfamily of receptors, is an important cutaneous sensor that detects thermal and chemical stimuli. TRPV3 is activated by innocuous warm temperature stimuli (>33° C) and a variety of physiologically active substances. While the corneal epithelium is known to respond to such stimuli, it is unknown whether TRPV3 is involved in this phenomenon. We show here that TRPV3 mRNA and protein are abundantly expressed in the epithelial cells of human and mouse cornea. Carvacrol, an agonist of TRPV3, elevated cytosolic Ca²⁺ concentration in both primary mouse corneal epithelial cells and cultured human corneal epithelial cells (HCE-T cells). The response to carvacrol was inhibited by ruthenium red, a TRPV channel antagonist. Moreover, repetitive agonist stimulation sensitized the response with gradually increasing amplitude, suggesting that the TRPV3 in the cornea has similar physiological and pharmacological characteristics to that in skin keratinocytes. Finally, a wound healing assay revealed that appropriate calcium ion influx via activated TRPV3 in corneal epithelial cells accelerated their proliferation. Thus, functional TRPV3 is present in corneal epithelial cells and may play a role not only in thermosensation, but also in the regulation of cell proliferation.     

Proliferation of retinal pigment epithelial cells induced by (R,R)-XY-10 and (S,S)-XY-10 and their action mechanisms

AIM: To investigate the mechanism of proliferation effect induced by (R,R)-XY-10 and (S,S)-XY-10 on retinal pigmented epithelial cells (ARPE-19). · METHODS: Human retinal pigmented epithelial cells (ARPE-19) and human umbilical vein endothelial cells (HUVECs) were used to investigate the effect of (R,R)-XY-10 and (S,S)-XY-10 on cell growth, and their mechanisms of proliferative action by using ERK, AKT, PI3K, Protein kinase C (PKC) and Nitric oxide synthase (NOS) inhibitors. · RESULTS: (R,R)-XY-10 and (S,S)-XY-10 dose-dependently increased ARPE-19 cell proliferation, but not on HUVECs. When treated with proliferative inhibitors, H-7 (5μmol/L), hypericin (20μmol/L), PD98059 (2μmol/L), LY294002 (50μmol/L), SH-5 (10μmol/L) and L-NAME (100μmol/L), the proliferative effect was reduced by H-7, hypericin, PD98059 and LY294002, but not by SH-5 and L-NAME. · CONCLUSION: (R,R)-XY-10 and (S,S)-XY-10 can induce cell proliferation through MAPK and PI3K dependent pathway.     

Epidermal growth factor receptor transactivation by the cannabinoid receptor (CB1) and transient receptor potential vanilloid 1 (TRPV1) induces differential responses in corneal epithelial cells

Corneal epithelial injury induces release of endogenous metabolites that are cannabinoid receptor 1 (CB1) and transient receptor potential vanilloid 1 (TRPV1) agonists. We determined the functional contributions by CB1 and TRPV1 activation to eliciting responses underlying wound healing in human corneal epithelial cells (HCEC). Both the selective CB1 and TRPV1 agonists (i.e., WIN55,212-2 [WIN] and capsaicin [CAP], respectively) induced EGFR phosphorylation whereas either inhibition of its tyrosine kinase activity with AG1478 or functional blockage eliminated this response. Furthermore, EGFR transactivation was abolished by inhibitors of proteolytic release of heparin bound EGF (HB-EGF). CB1-induced Ca²⁺ transients were reduced during exposure to either the CB1 antagonist, AM251 or AG1478. Both CAP and WIN induced transient increases in Erk1/2, p38, JNK1/2 MAPK and Akt/PI-3K phosphorylation status resulting in cell proliferation and migration increases which mirrored those elicited by EGF. Neither EGF nor WIN induced any increases in IL-6 and IL-8 release. On the other hand, CAP-induced 3- and 6-fold increases, which were fully attenuated during exposure to CPZ, but AG1478 only suppressed them by 21%. The mixed CB1 and TRPV1 antagonist, AM251, enhanced the CAP-induced rise in IL-8 release to a higher level than that elicited by CAP alone. In conclusion, CB1 and TRPV1 activation induces increases in HCEC proliferation and migration through EGFR transactivation leading to global MAPK and Akt/PI-3K pathway stimulation. On the other hand, the TRPV1-mediated increases in IL-6 and IL-8 release are elicited through both EGFR dependent and EGFR-independent signaling pathways.     

重组人表皮生长因子治疗外伤性角膜上皮缺损

目的　评价重组人表皮生长因子 (rhEGF)滴眼液治疗外伤性角膜上皮缺损的临床疗效。方法　观察 84例外伤性角膜上皮缺损病例 ,随机分成rhEGF滴眼液治疗组和常规治疗对照组 (0 3 %氧氟沙星滴眼液 )各 42例。治疗组用rhEGF滴眼液 ,对照组用 0 3 %氧氟沙星滴眼液 ,每日滴眼 4次 ,共 10天。隔日观察上皮缺损修复情况。结果　rhEGF治疗外伤性角膜上皮缺损的有效率明显高于常规治疗对照组 ,两者差异有非常显著意义 (P <0 0 1)。结论　rhEGF滴眼液治疗外伤性角膜上皮缺损能有效地促进角膜上皮缺损的修复。     

Regulation of thioredoxin by ceramide in retinal pigment epithelial cells

The purpose of this study was to determine the expression, regulation and signaling of a key redoxin family member thioredoxin 1 (Trx1) in normal, oxidant-stimulated and growth factor-pretreated RPE cells. Trx1 is expressed in early passage, human RPE cell cultures. RPE cells exposed to C₂-ceramide for 24 h showed no significant change in expression of Trx1 vs. controls with and without pretreatment for 24 h with hepatocyte growth factor (HGF). Neither hypoxia from 1% O₂ or from CoCl₂ exposure resulted in any alteration in Trx1 expression in RPE cells. C₂-ceramide treatment caused translocation of Trx1 from cytosol to the nucleus, which was abolished by pre-treatment of cells with a p38 MAPK-specific inhibitor. Furthermore, the gene and protein expression of thioredoxin interacting protein (Txnip) increased with ceramide treatment and was significantly (p < 0.001) elevated with HGF preincubation vs. untreated controls. Prominent protection from ceramide-induced RPE cell death by exogenous rTrx1 was demonstrated. Although Trx1 directly interacts with its inhibitor, Txnip, p38 inhibition does not appear to have a role in this interaction. We found no direct interaction between apoptosis signal regulating kinase (ASK-1) and Txnip under the same experimental conditions. In summary, our data demonstrate the expression of Trx1 and Txnip in human RPE cells. Ceramide treatment results in translocation of Trx1 to the nucleus, and upregulation of Txnip expression; exogenous rTrx1 protects from ceramide-induced cell death. These results suggest that Trx1 and Txnip play an important role in the response of RPE to ceramide toxicity.     

Inhibition of corneal neovascularization by recombinant adenovirus mediated antisense VEGF RNA

The expression of vascular endothelial growth factor has been strongly implicated in the pathogenesis of conditions leading to inappropriate blood vessel growth in the eye. As such, vascular endothelial growth factor is an attractive target for anti-angiogenic therapies designed to treat neovascular eye diseases. One such therapy, antisense gene therapy, is a technique based on the ability of single-stranded DNA or RNA sequences to alter the expression of targeted genes. Recombinant adenoviruses have demonstrated efficient ocular cell transduction with a high level of transgene production. Cauterization of the normally avascular rat cornea results in a strong neovascular response, making it an ideal animal model for the testing of anti-angiogenic therapies. In this study, a recombinant adenovirus system was assessed for the ability to express biologically relevant antisense RNA to reduce vascular endothelial growth factor expression in a rat model of corneal neovascularization. Recombinant adenovirus constructs expressing short and long antisense and sense vascular endothelial growth factor cDNA, under the control of cytomegalovirus major immediate early promoter or the RNA polymerase III promoter, VA1, were constructed. The expression of short and long antisense RNAs was demonstrated by Northern blot hybridization. All constructs were capable of producing RNA, and the highest level of antisense RNA production was detected in retinal pigment epithelial cells which had been transduced with the longer antisense cDNA construct under the control of the VA1 promoter. This construct was also the most efficient in reducing in vitro vascular endothelial growth factor production (P<0.05) and human endothelial cell proliferation. This construct was subsequently injected into rat eyes 24hr prior to cauterization of the cornea and antisense vascular endothelial growth factor expression was demonstrated by in situ hybridization. The resulting neovascular response was clearly inhibited at 4, 7 and 14 days post-cautery, compared to the control injections which demonstrated an intense neovascular response. Only one out of six eyes injected with the long antisense cDNA construct under the control of the VA1 promoter demonstrated any vascular response to cautery. The reduction in the neovascular response was correlated, with significantly lower amounts of vascular endothelial growth factor protein in the corneas (P=0.006). These observations suggest that the specific down-regulation of vascular endothelial growth factor production is sufficient to reduce the corneal neovascular response and that recombinant adenovirus might be a useful vehicle to produce antisense RNA in situ to down-regulate ocular gene expression.     

Effects of epidermal growth factor on transforming growth factor-beta1-induced epithelial-mesenchymal transition and potential mechanism in human corneal epithelial cells

AIM: To evaluate the effects of epidermal growth factor (EGF) on transforming growth factor-beta1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) in human corneal epithelial cells (HCECs). METHODS: HCECs were cultured and treated with TGF-β1 for establishing the model of EMT in vitro. Biological effect of EGF on TGF-β1-induced EMT was evaluated. Proteins and mRNAs expression changes of E-cadherin, N-cadherin and Fibronectin (EMT-relative markers) after TGF-β1 or TGF-β1 combined EGF treatment were detected by Western blot and RT-PCR, respectively. Viability and migration of HCECs were measured by CCK-8, transwell cell migration assay and cell scratch wound healing assay. Activation of Smad2, ERK, p38, JNK and Akt signaling pathways were evaluated by Western blot. Inhibitors of relevant signaling pathways were added to the HCECs to explore the key signal mechanism. RESULTS: With treatment of TGF-β1 only, three EMT-relative proteins and mRNA expression showed that EMT up-regulated in a concentration-dependent and time-dependent manner, with significantly decreasing cell viability (TGF-β1≥5 ng/mL, P<0.05) and increasing cell migration (TGF-β1≥5 ng/mL, P<0.01). The phosphorylation of Smad2 and p38 was a key process of TGF-β1-induced EMT. Meanwhile, EMT-relative proteins and mRNA expression showed that EGF inhibited TGF-β1-indued EMT, with significantly increasing cell viability (EGF≥10 ng/mL, P<0.01). It was noteworthy that EGF significantly enhanced cell migration although EMT was inhibited (EGF≥10 ng/mL, P<0.01), and the blockage of p38 (by SB202190, a p38 inhibitor) was a potential mechanism of this phenomenon. CONCLUSION: EGF inhibits TGF-β1-induced EMT via suppressive p38, and promotes cells proliferation and migration in a non-EMT process by inhibiting p38 pathway.