角质细胞生长因子2促进兔角膜碱烧伤上皮愈合机制的研究

目的　探讨角质细胞生长因子2 (KGF- 2 )对实验性兔角膜中央碱烧伤后角膜上皮愈合的作用及其机制。方法　24只新西兰白兔的24只角膜碱烧伤眼按随机数字法分成4组,每组6只眼,其中A、B、C组为治疗组,分别以3种不同浓度: 1μg/ml、50μg/ml、100μg/mlKGF 2滴眼液治疗;D组为对照组,用磷酸盐缓冲液(PBS)滴眼液治疗;观察角膜上皮愈合情况,并做形态学检查及P63、角质蛋白单克隆抗体(AE5)、表皮细胞生长因子单克隆抗体(EGFR)免疫组化研究。结果　1 ~100μg/mlKGF 2能够促进兔角膜上皮愈合。角膜碱烧伤24h后100μg/mlKGF- 2组和对照组角膜上皮愈合率分别为40%和74% (P<0 .05);第4天时4组均出现一定反复;第10天时各治疗组近完全愈合。碱烧伤后第7天即可观察到P63阳性细胞不仅存在于角膜缘区的部分基底细胞中,同时也向角膜内迁移。如角膜碱烧伤后第7天角膜缘区P63阳性细胞数: 100μg/mlKGF 2组为( 53 .8±2. 6)个,对照组为(29. 5±2. 2)个,正常角膜为(17. 0±2. 1)个(P=0. 000);同时非角膜缘区P63阳性细胞数分别为: 100μg/mlKGF 2组为(69. 5±2. 8)个,对照组为(19 5±2 8)个,正常角膜为0个(P=0 .000)。结论　KGF- 2可激活角膜上皮干细胞,使其不断增殖分化,从而促进角膜上皮损伤的愈合。     

生长因子对角膜上皮细胞损伤修复的作用

角膜上皮细胞是维持角膜正常生理功能的重要细胞。角膜上皮缺损 ,一般修复较快 ,但也有些角膜疾患可导致持续性角膜上皮缺损 ,从而引起一系列并发症。一些生长因子可促进角膜上皮的修复 ,开创了治疗角膜上皮持续缺损的新方法。本文着重介绍与角膜上皮细胞关系密切的表皮生长因子 (EGF)、血小板源性生长因子 (PDGF)、成纤维细胞生长因子 (FGF)、转化生长因子 (TGF)、肝细胞生长因子 (HGF)和角质细胞生长因子 (KGF)的结构及其在角膜上皮细胞修复中的作用。     

维生素A棕榈酸酯对兔机械性角膜上皮损伤愈合及结膜杯状细胞的作用研究

目的 探讨维生素A棕榈酸酯与重组牛碱性成纤维细胞生长因子对兔机械性角膜上皮损伤愈合及结膜上皮细胞、杯状细胞的作用.方法 实验研究.选取雄性新西兰大白兔120只,建立机械性角膜上皮损伤模型(角膜中央直径8 mm上皮刮除),随机数字表法分为4组,每组30只.A组使用盐酸林可霉素滴眼液,B组合用维生素A棕榈酸酯凝胶与盐酸林可霉素滴眼液,C组合用重组牛碱性成纤维细胞生长因子凝胶与盐酸林可霉素滴眼液,D组合用重组牛碱性成纤维细胞生长因子凝胶与维生素A棕榈酸酯凝胶及盐酸林可霉素滴眼液.各组均于机械性角膜上皮损伤建模后当日开始用药.用法均为3次/d,1滴/次,观察时间为7 d.在模型建立第0、1、4、7天共4个时间点采用前节裂隙灯显微镜照相系统进行眼表照相,并计算角膜上皮损伤及其修复面积;在模型建立前及建立后第1、4、7天共4个时间点进行角结膜组织透射电镜检查、角膜光镜组织学检查及结膜印迹细胞学检查,对角膜上皮、结膜上皮细胞修复情况及结膜杯状细胞形态数量进行分析.采用方差分析、Tukey显著性检验等对数据进行统计学分析.结果 造模后第1天各组角膜上皮已愈合面积比较差异有统计学意义(F=17.663,P=0.000),角膜上皮已愈合面积A组(53.512±18.850)mm~2,B组(92.194±14.367)mm~2,C组(89.779±20.535)mm~2,D组(127.816±16.379)mm~2,B组与C组之间差异无统计学意义(P=0.995);结膜印迹细胞学检查显示,各组结膜杯状细胞数量(每740μm×550μm 面积)明显下降,A组(10.083±4.441)个,B组(10.667±3.551)个,C组(9.583±4.502)个,D组(9.167±5.606)个,但各组之间的差异无统计学意义(F=0.239,P=0.868).造模后第4天各组角膜上皮已愈合面积比较差异有统计学意义(F=37.665,P=0.000),角膜上皮已愈合面积A组(120.369±11.839)mm~2,B组(156.606±8.087)mm~2,C组(154.216±9.990)mm~2,D组(175.181±5.168)mm~2,B组与C组之间差异无统计学意义(P=0.968);结膜印迹细胞学检查显示,各组结膜杯状细胞数量(每740 μm ×550 μm 面积)开始恢复,A组(41.250±4.575)个,B组(56.083±6.374)个,C组(48.417±4.562)个,D组(61.917±5.017)个,各组之间比较差异有统计学意义(F=36.210,P=0.000).造模后第7天各组角膜上皮已愈合面积A组(177.472±3.585)mm~2,B组(186.715±3.022)mm~2,C组(182.293±3.158)mm~2,D组(194.106±2.176)mm~2,D组角膜上皮已愈合面积大于其他3组(P<0.05);结膜印迹细胞学检查显示,各组结膜杯状细胞数量(每 740 μm×550 μm面积)明显恢复,A组(63.167±11.488)个,B组(99.501±15.877)个,C组(82.015±9.175)个,D组(104.750±9.659)个,各组之间比较差异有统计学意义(F=30.312,P=0.000),B组与D组之间比较差异无统计学意义(P=0.700).透射电镜下观察角膜组织,维生素A棕榈酸酯和重组牛碱性成纤维细胞生长因子均能促进角膜上皮细胞之间连接的建立,维生素A棕榈酸酯具有保护角膜上皮细胞、防止上皮角化、促进角膜上皮增殖分化的作用.电镜检查结膜组织,可见B、D组新生的结膜杯状细胞数量较多,胞内分泌颗粒密集,与A、C组有明显差异,B、C、D组新生的结膜上皮细胞连接紧密.结论 维生素A棕榈酸酯和重组牛碱性成纤维细胞生长因子均能够有效促进机械性角膜上皮损伤的角膜上皮修复,合并用药时作用最明显.维生素A棕榈酸酯可促进结膜杯状细胞的再生,促进结膜上皮细胞间连接的建立,明显优于重组牛碱性成纤维细胞生长因子.     

KGF-2治疗兔角膜中央碱烧伤的实验研究

目的　观察角质细胞生长因子 2 (KGF 2 )对实验性兔角膜中央碱烧伤的治疗效果。方法　将 2 4只兔角膜碱烧伤眼随机分成 4组 ,用KGF 2滴眼液治疗 ,以后行荧光素染色裂隙灯显微镜照相 ,照片经计算机图象系统分析 ,角膜切片HE染色 ,光镜检查。结果　 10 0 μg/mLKGF 2角膜上皮愈合率最高 ,且与剂量呈依赖性 ,各治疗组与对照组之间差异均有显著性意义 ,P <0 0 5。结论　KGF 2能够促进兔角膜碱烧伤上皮的愈合     

PRK后角膜TGF—β1mRNA的表达与Haze形成

目的 :为研究角膜上皮下混浊 ( haze)形成的发生机理 ,检测激光角膜光学切除术 ( photorefractive keratectomy,PRK)后角膜上皮和基质表达转移生长因子 - β1 ( transforming growth factor- β1 ,TGF- β1 )的变化。方法 :新西兰白兔 2 0只 ,随机分成 4组 ,其中 15只施行 PRK。于术后 1、2、3月用裂隙灯显微镜观察 haze形成情况 ,并用原位核酸分子杂交方法 ,检测角膜上皮和基质 TGF- β1 m RNA的表达。结果 :PRK后 1月已有 haze形成 ,2月时 haze最明显 ,术后 3月 haze减轻或消失。正常角膜上皮和基质有 TGF- β1 m RNA表达 ,PRK后角膜表达 TGF- β1 m RNA增加 ,且术后 2月表达最强。 TGF- β1 m RNA表达强弱与形成 haze的轻重密切相关。结论 :TGF- β1 参与 PRK后伤口愈合过程 ,且调节着 haze的形成和发展     

穿透性角膜移植术后早期上皮愈合的影响因素分析

目的　分析穿透性角膜移植术后早期植片上皮缺损和愈合的相关影响因素。方法　 61例 62眼行部份穿透性角膜移植手术 ,裂隙灯显微镜和荧光素染色法观察术后植片上皮的缺损情况 ,分类并记录植片上皮的愈合时间 ,分析植片上皮愈合时间与角膜原发疾病、新生血管增生、受体性别、年龄以及供体眼球保存时间的关系。结果　术后植片上皮的平均愈合时间为 3 45天± 2 72天 ,圆锥角膜组的植片上皮愈合时间与大泡性角膜病变组、角膜白斑组相比明显缩短 ,差异有统计学意义(P值为 :0 0 45和 0 0 0 7) ;植片上皮愈合时间与受体性别、植床新生血管增生无关 (P值均 >0 0 5 ) ;大于 61岁年龄组与小于 2 0岁年龄组、 2 0岁至 40岁年龄组的上皮愈合时间相比明显延长 ,差异有统计学意义 (P值为 0 0 0 3和 0 0 0 4) ;供体眼球保存时间大于 12小时组的植片上皮愈合时间较小于 12小时组明显延长 ,差异具有统计学意义 (P值 =0 0 0 8)。结论　植片上皮愈合时间受原发角膜疾病种类、受体年龄、及供体眼球保存时间的影响 ,与受体性别、植床新生血管增生情况无关。     

唯地息眼凝胶剂治疗兔芥子气角膜损伤

目的:观察唯地息眼凝胶剂对兔芥子气角膜上皮损伤的治疗作用。方法:新西兰白兔8只,用200μL/L液态芥子气致伤双眼角膜5min,左眼为治疗组,用唯地息眼凝胶剂点眼,4次/d,右眼为对照组,用2.5g/L氯霉素眼液滴眼,4次/d,分别于染毒后2,8,16,24,36,48和72h对角膜荧光素着色区拍照,计算其上皮愈合速率和上皮破损率。结果:治疗组和对照组的角膜上皮愈合速率分别为(1.15±0.19)mm2/h和(1.03±0.12)mm2/h,两者有显著差异(P<0.05),角膜上皮破损率分别为44.4%和54.2%,两者差异显著(P<0.05)。结论:唯地息眼凝胶剂能促进芥子气染毒引起的兔眼角膜损伤的上皮愈合,也能减少角膜上皮反复破损。     

肝细胞生长因子对视网膜色素上皮细胞增生及损伤愈合的影响

目的 研究肝细胞生长因子(hepatocyte growth factor，HGF)对培养的人视网膜色素上皮(retinal pigmental epithelial，RPE)细胞增生和损伤愈合的影响。方法 MTT比色法检测细胞增生情况，相差显微和细胞计数法观察RPE细胞损伤后的愈合情况。结果 中等浓度HGF(10ng／ml)刺激RPE细胞的增生较低浓度组(0．01～1ng／m1)明显增多(P＜0．05)，而与高浓度(100ng／ml)组比较无统计学差异(P＞0．05)。随着HGF刺激时间的延长，迁移的细胞数量增多，同一时间点，HGF处理组明显比对照组细胞数量多(P＜0．05)。结论 中等浓度的HGF可促进细胞增生，对损伤后的RPE细胞有迁移作用。     

角膜缘niche细胞与基质细胞维持角膜缘干细胞功能的对比研究

目的比较角膜缘niche细胞(limbal niche cells,LNCs)与角膜缘基质细胞(limbal stromal cells,LSCs)在维持角膜缘干细胞功能上的不同特性。方法将LNCs和LSCs分别从6个角膜缘组织分离,并在相同的条件下培养、传代。LNCs与LSCs经丝裂霉素C(mitomycin C,MMC)处理后分为LNCs组与LSCs组作为饲养细胞分别与角膜缘干细胞共培养,比较两组角膜缘干细胞克隆形成率(colony-forming efficiency,CFE)、上皮细胞复层化以及细胞标志物和部分基因的表达。结果 LNCs组角膜缘干细胞CFE(6.57±1.54)%高于LSCs组(1.43±0.47)%。LNCs组细胞复层上皮数(4～5层)多于LSCs组(2～3层)。角膜缘干细胞克隆与免疫荧光染色及mRNA半定量分析结果显示,LNCs组比LSCs组表达了更多干细胞标志物ΔNp63,能更有效地维持角膜缘干细胞的细胞特性。逆转录PCR分析结果显示,LNCs组与LSCs组都分泌了一些维持角膜缘干细胞生长的生长因子,但LNCs组比LSCs组高表达上皮型钙黏蛋白(E-cad...     

大鼠烟曲霉菌性角膜炎初期核苷酸结合寡聚域样受体在角膜中的表达及其免疫防御作用

背景核苷酸结合寡聚域（NOD）样受体（NLRs）在天然免疫过程中发挥重要作用,但其在真菌性角膜炎发生和发展过程中的作用研究少见。目的研究NOD,在大鼠烟曲霉菌性角膜炎（AFK）角膜组织中的表达及作用。方法72只成年清洁级Wistar大鼠按照随机数字表法随机分为正常对照组、单纯角膜上皮损伤组和AFK模型组,均以右眼作为实验眼。正常对照组12只大鼠中选取6只仅刮取角膜上皮用于逆转录PCR（RT—PCR）实验,另6只大鼠制备角膜标本用于组织病理学检查、免疫组织化学和免疫荧光染色实验。单纯角膜上皮损伤组共30只大鼠,仅刮除中央角膜上皮,AFK模型组大鼠共30只,刮除角膜上皮后接种烟曲霉菌标准株。分别于实验后4、8、16、24h选取各组6只大鼠刮取角膜上皮,采用RT—PCR法检测角膜中NOD2mRNA的表达,其他6只于实验后24h制备角膜标本,采用免疫组织化学法和免疫荧光染色法检测角膜中NOD2蛋白的表达,并将各组检测结果进行比较。实验后4、8、16、24h分别摘除各组大鼠右眼眼球,制作角膜标本进行常规组织病理学检查。结果裂隙灯显微镜下观察各组大鼠各时间点的角膜情况,均造模成功。RT—PCR结果显示,正常对照组角膜仅有微量NOD2mRNA的表达,单纯角膜上皮损伤组和AFK模型组大鼠角膜中NOD2mRNA的相对表达量明显增加,且在实验4、8、16、24h,AFK模型组大鼠角膜中NOD2mRNA的相对表达量均明显高于单纯角膜上皮损伤组,差异均有统计学意义（t=-0．409、-0．439、-0．534、-0．618,均P=0．000）。大鼠角膜组织病理学检查显示,正常对照组大鼠角膜结构完整;单纯角膜上皮损伤组可见到少部分角膜上皮缺损、前弹力层皱褶及角膜轻度水肿,有少量中性粒细胞浸润;AFK模型组大鼠可见角膜溃疡,角膜水肿增厚,浅基质层可见大量中性粒细胞浸润。     

Delay of corneal epithelial wound healing and induction of keratocyte apoptosis by platelet-activating factor

PURPOSE: To examine the role of the lipid mediator platelet-activating factor (PAF) in epithelial wound healing. METHODS: A 7-mm central de-epithelializing wound was produced in rabbit corneas, and the tissue was incubated with 125 nM carbamyl PAF (cPAF), an analogue of PAF. Rabbit corneal epithelial and stromal cells were also cultured in the presence of cPAF. Cell adhesion, proliferation, and migration assays were conducted. Apoptosis was assayed by TUNEL staining on preparations of corneal tissue sections and in cells in culture. RESULTS: Twenty-four hours after injury, 50% of the wounded area was covered by new epithelium, whereas only 30% was covered in the presence of cPAF. At 48 hours, the epithelium completely closed the wound, but only 45% of the original wound was covered in corneas treated with cPAF. Similar inhibition of epithelial wound closure was found with human corneas incubated with PAF in organ culture. Moreover, addition of several growth factors involved in corneal wound healing, such as epidermal growth factor, hepatocyte growth factor, and keratinocyte growth factor, could not overcome the inhibitory action of PAF in wound closure. Three PAF antagonists, BN50727, BN50730, and BN50739, abolished the effect of PAF. A significant increase in TUNEL-positive staining occurred in corneal stromal cells (keratocytes), which was inhibited by preincubating the corneas with PAF antagonists. However, no TUNEL-positive staining was found in epithelial cells. TUNEL-staining results in cultured stromal cells (keratocytes) and epithelial cells in first-passage cell culture were similar to those in organ-cultured corneas. In addition, PAF caused 35% to 56% inhibition of adhesion of epithelial cells to proteins of the extracellular matrix: collagen I and IV, fibronectin, and laminin. There were no significant changes in proliferation or migration of epithelial cells induced by the lipid mediator. CONCLUSIONS: The results suggest PAF plays an important role in preventing corneal wound healing by affecting adhesion of epithelial cells and increasing apoptosis in stromal cells. PAF antagonists could be of therapeutic importance during prolonged ocular inflammation, helping to avoid loss of corneal transparency and visual acuity.     

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.     

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.     

角质细胞生长因子促进角膜上皮损伤修复的研究

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方法。方法用３Ｈ－胸腺嘧啶核苷（３Ｈ－ＴｄＲ）掺入及液体闪烁技术，观察不同浓度的外源性角质细胞生长因子（ｋｅｒａｔｉｎｏｃｙｔｅｇｒｏｗｔｈｆａｃｔｏｒ，ＫＧＦ）对体外培养的人角膜上皮细胞生长的影响，由此推算出有效滴眼液浓度并应用于兔眼。用计算机图形分析系统计算角膜上皮愈合速率；用光镜和电镜评估愈合的质量。结果０．１～１００ｎｇ／ｍｌＫＧＦ有明显促进体外培养的人角膜上皮细胞生长的作用（增长率为２７．６６％～７６．７３％），且呈剂量依赖性（ｒ＝０．９２３３，Ｐ＜０．００１）。１μｇ／ｍｌＫＧＦ滴兔眼，加速了角膜上皮损伤修复（愈合速率，ＫＧＦ组为１．７７±０．２３ｍｍ２／ｈ，与对照组１．４９±０．２４ｍｍ２／ｈ比较，Ｐ＜０．０５）。结论外源性ＫＧＦ对体外培养的人角膜上皮细胞有明显的促生长作用，其滴眼液有加速兔眼角膜上皮创伤修复的作用。     

HGF- and KGF-induced activation of PI-3K/p70 s6 kinase pathway in corneal epithelial cells: its relevance in wound healing.

In this study we have investigated the involvement of PI-3K and its downstream target p70 S6K in the signaling response of corneal epithelial cells after HGF and KGF stimulation. HGF induced three- to five-fold increase in PI-3K activity in 5-10 min, whereas KGF stimulation resulted in two- to three-fold increase in activity in 2-10 min. Both growth factors also caused the phosphorylation of p70 S6K and stimulation of its activity. HGF increased p70 S6K activity by 300% and KGF by about 200%. Protein kinase C (PKC) activator TPA also induced the phosphorylation of p70 S6K. Both the PI-3K inhibitor wortmannin and PKC inhibitor calphostin C blocked the phosphorylation of p70 S6K mediated by the growth factors. However, the mitogen-activated protein kinase (p42/44 MAPK) cascade inhibitor PD98059 had no effect on p70 S6K activation. Furthermore, HGF and KGF increased the rate of corneal epithelial wound healing in an organ culture model, and wortmannin and rapamycin (the p70 S6K inhibitor) blocked corneal epithelial wound healing promoted by the growth factors. These studies suggest that PI-3K and p70 S6K are important signal transducers in the stimulation of corneal epithelial cells by HGF and KGF. PKC is involved in the PI-3K-dependent activation of p70 S6K but not MAPK. Inhibition of wound closure by PI-3K and p70 S6K inhibitors suggests these enzymes play a significant role in corneal wound repair stimulated by HGF and KGF.     

Phosphorylation of MAP kinase in corneal epithelial cells during wound healing

PURPOSE: To investigate the role of mitogen-activated protein kinase (MAPK), such as p44/42 MAPK, p38 MAPK and stress-activated protein kinase (SAPK), in corneal epithelial cells during the wound healing process. METHODS: A single non-penetrating incision was produced on rat cornea. Then the corneal wound healing process was observed with an immunocytochemical technique using specific antibodies reacting only with phosphorylated p44/42 MAPK, p38 MAPK or SAPK. Cell lysates of corneal epithelial cells in rabbits stimulated with keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) were processed for Western blot using antibodies to phosphorylated p44/42 MAPK. RESULTS: Maximum activation of p44/42 MAPK was observed in wing and basal cells at wounded regions in rat cornea at 1 hour after the incision. Activation of p44/42 MAPK was still detected in all basal and wing cells at wounded regions at up to 24 hours when the incisions were completely closed, and then receded to normal intensity after 7 days. Neither p38 MAPK nor SAPK were activated during the wound healing process. Western blot analysis of cultured corneal epithelial cells in rabbits showed phosphorylation of p44/42 MAPK after 30 minutes in response to KGF and HGF, whereas non-activated p44/42 MAPK was ordinarily detected even at the absence of KGF or HGF. CONCLUSIONS: These results demonstrate that p44/42 MAPK is activated during the corneal wound healing process and suggest that KGF and HGF play an important role in initiation of cell migration and proliferation in the initial wound healing process by activating p44/42 MAPK.     

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.     

Platelet-activating factor (PAF) enhances apoptosis induced by ultraviolet radiation in corneal epithelial cells through cytochrome c-caspase activation

PURPOSE: To examine the role of platelet-activating factor (PAF) on apoptosis of corneal epithelial cells exposed to radiation. METHODS: Rabbit corneal epithelial (RCE) and human corneal epithelial (HCE) cells were exposed to UVC radiation and then to carbamyl PAF (cPAF) for different increments of time. The PAF antagonist BN50739 was added 30 min before cPAF. The caspase inhibitors Ac-DEVD-CHO and Ac-YVAD-CHO were added 1 h before, and the phospholipase A(2) (PLA(2)) inhibitor MAFP was added 3 h before UVC irradiation. FITC-dUTP TUNEL and DAPI staining were performed to assess the percentage of apoptotic cells. DNA ladder analysis was used to investigate apoptosis induced by different intensities of UVC (50-600 J/m(2)) with or without cPAF. Caspase activation and release of cytochrome c from mitochondria to cytosol were determined by Western blot. RESULTS: While only 2.7% of RCE cells were DAPI positive in controls incubated for 12 h, 44% of cells were stained positive 4 h after irradiation; these values increased to 63% in the presence of cPAF. Cells incubated with cPAF alone were similar to controls. TUNEL staining and DNA laddering showed also increased in apoptosis after PAF treatment of UV-irradiated cells and BN50739 blocked the effect of cPAF. cPAF increased caspase-3 activation induced by UV irradiation in HCE cells. Cytochrome c release from mitochondria to cytosol was observed 30 min after irradiation. cPAF almost doubled the release of cytochrome c at 30 min and 1 h. Here, too, BN50739 blocked the PAF effect. No release of cytochrome c by PAF was seen in non-irradiated cells, even at higher concentrations. MAFP caused a decrease in cytochrome c release from UV-treated cells, and caused an even greater inhibition of cytochrome c release in cells stimulated with PAF. CONCLUSIONS: PAF increases RCE and HCE apoptosis caused by UV irradiation by stimulating PLA(2), producing an early release of cytocrome c from mitochondria and activating caspase-3 by a receptor-mediated mechanism. This accelerating effect of PAF on the apoptotic cascade only occurred when corneal epithelial cells had been previously damaged by UV radiation.     

Stromal-epithelial interactions in the cornea

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