HSV-tk/GCV系统对人Tenon囊成纤维细胞的旁观者效应及其机制研究

目的观察HSV—tk／GCV系统对人Tenon囊成纤维细胞（HTFs）的旁观者效应并研究其作用机制。方法将转HSV—tk基因HTFs（HTFs—tk）与正常HTFs于两种接种密度下以不同比例混合，加入5μg／ml更昔洛韦作用5d，MTT法检测对HTFs的杀伤作用；染料传输法观察HTFs细胞间隙连接细胞间通讯（GJIC）的能力；免疫细胞化学检测Cx43分布；RT—PCR及Westernblot检测Cx43表达。结果旁观者效应随HTFs—tk／HTFs比例增加而增强，高细胞接种密度组明显强于低密度组；染料通过GJIC传输6级以上；Cx43主要分布于HTFs细胞膜并检测到其mRNA与蛋白表达。结论旁观者效应需要细胞间密切接触，Cx43介导GJIC可能是HSV—TK／GCV系统对HTFs产生旁观者效应的主要机制，HTFs表达Cx43可能在结膜伤口愈合反应中发挥重要作用。     

氯化锂对人Tenon囊成纤维细胞增生抑制作用的机制研究

目的:探讨氯化锂对人Tenon囊成纤维细胞(HTFs)缝隙连接细胞间通讯(GJIC)的影响及其可能的作用机制。方法:收集2019年4月于德州市人民医院眼科行斜视手术的1例患者的Tenon囊组织,剪成1 mm×1 mm×1 mm的组织块,进行原代培养并传代,取第4代HTFs进行实验。将HTFs分为对照组和氯化锂处理组,对...     

结缔组织生长因子对人Tenon囊成纤维细胞中E-cadherin蛋白表达的促进作用

背景 青光眼滤过手术后滤过通道的瘢痕化是手术失败的主要原因,其主要病理机制是成纤维细胞的异常增生、间质-上皮转分化及细胞外基质的重塑.结缔组织生长因子(CTGF)是促进瘢痕形成的关键因子,而CTGF是否能促进人Tenon囊成纤维细胞(HTFs)的间质-上皮转分化尚不清楚.目的 观察CTGF对HTFs间质-上皮转分化的影响.方法 用体积分数10％胎牛血清的DMEM高糖型培养液进行HTFs体外常规培养和传代,取3～6代细胞用于实验.将培养的HTFs分为空白对照组和CTGF处理组,空白对照组用DMEM完全培养液培养细胞,CTGF处理组在培养液中加入CTGF,使终质量浓度为50 ng/ml.细胞培养后48 h,采用细胞免疫荧光染色技术鉴定HTFs中上皮钙黏素(E-cadherin)蛋白的表达,采用Western blot法对HTFs中E-cadherin蛋白的表达量进行检测.结果 空白对照组及CTGF处理组HTFs均生长良好,呈长梭形,漩涡状排列.细胞免疫荧光染色显示,CTGF处理组HTFs细胞质呈红色荧光,细胞核呈蓝色荧光;空白对照组HTFs仅见DAPI蓝染的细胞核,无E-cadherin表达的红色荧光.Western blot法检测结果显示,空白对照组HTFs中E-cadherin蛋白无表达,而CTGF处理组HTFs中E-cadherin蛋白的相对表达量为0.63±0.08.结论 间叶组织来源的成纤维细胞本身不表达E-cadherin,在CTGF的刺激下成纤维细胞能够表达E-cadherin,CTGF促进HTFs的间质-上皮转分化.     

miR-200a对结膜成纤维细胞增生和迁移的调控作用及其机制

背景 青光眼滤过性手术后手术区域瘢痕化是导致抗青光眼手术失败的主要因素,因此越来越多的研究关注瘢痕形成的原因和过程. 目的 比较青光眼滤过术后滤过区人瘢痕组织来源成纤维细胞(HSFs)与人正常Tenon囊来源成纤维细胞(HTFs)增生迁移能力的差异以及微小RNA200a(miR-200a)对结膜成纤维细胞生物学行为的抑制作用,并探讨产生这种差异的可能机制. 方法 收集斜视手术过程中切取的正常Tenon囊组织和青光眼滤过术后手术区瘢痕组织,对成纤维细胞进行原代培养,采用细胞计数试剂盒8(CCK8)法检测不同来源成纤维细胞的增生能力;采用细胞划痕试验检测细胞的相对迁移距离;采用实时荧光定量PCR法检测不同来源成纤维细胞中转化生长因子-B1(TGF-β1)mRNA和miR-200a mRNA的表达.于HTFs培养基中分别加入0、1、2和5 ng/ml TGF-β1拟似物,于HSFs培养基中分别加入0.00、0.25、0.50、1.00μs/mlTGF-β1抑制剂,细胞处理24 h,采用CCK8法检测细胞增生能力;用2 ng/ml TGF-β1拟似物处理HTFs,用1.00 μg/ml TGF-β1抑制剂处理HSFs,分别采用划痕法检测不同处理组细胞的相对迁移距离,采用实时荧光定量PCR法检测不同处理组细胞中miR-200a mRNA的相对表达量. 结果 原代培养的细胞胞体较大,呈纺锤形或星形,细胞核呈卵圆形,对角蛋白抗体呈弱阳性反应,对波形蛋白抗体呈阳性反应.HSFs的增生值(A值)为1.476±0.110,明显高于HTFs的0.958±0.074,差异有统计学意义(t=24.900,P=0.016);HSFs中TGF-β1 mRNA相对表达量高于HTFs,是HTFs的(1.739±0.205)倍,差异有统计学意义(t=6.358,P=0.024);HSFs中miR-200a mRNA的相对表达量明显低于HTFs,是HTFs的(46.23±10.78)％,差异有统计学意义(t=7.394,P=0.018).与添加2 ng/ml TGF-β1拟似物的HTFs相比,添加TGF-β1拟似物的HSFs相对迁移距离增加了(3.533±0.402)倍,miR-200a mRNA相对表达量低至(45.4±7.3)％,差异均有统计学意义(均P＜0.05);与添加1.00 μg/ml TGF-β1抑制剂的HTFs相比,培养基中添加TGF-β1抑制剂后,HSFs的相对迁移距离幅度降低至(64.3±6.5)％,miR-200a mRNA相对表达量明显增加(3.106±0.415)倍,差异均有统计学意义(均P＜0.05).结论 HSFs的增生及迁移能力均明显强于HTFs,可能与HSFs中miR-200a表达量低有关.MiR-200a与TGF-β1在调控成纤维细胞的增生和迁移能力方面发挥相反的作用.     

EGF对人视网膜色素上皮细胞胞间通讯功能和Cx43表达的影响

目的探讨培养的人视网膜色素上皮(retinalpigmentepithelium,RPE)细胞中连接蛋白Cx43的表达,以及表皮生长因子(epidermalgrowthfactor,EGF)对人视网膜色素上皮细胞间缝隙连接功能、细胞间通道蛋白Cx43表达的影响。方法免疫组化的方法观察Cx43在培养的人RPE细胞中的表达特点,用10mg·L-1、20mg·L-1、30mg·L-1不同浓度的EGF作用于培养的第4代人RPE细胞后,观察缝隙连接蛋白Cx43的表达强弱变化,用计算机灰度测量的方法评估表达的强度并作统计学分析。用LY染料传输方法测定细胞间隙连接功能,观察这3组不同浓度的EGF对人RPE细胞缝隙连接功能的影响。结果免疫组化染色显示体外培养的人RPE细胞表达缝隙连接蛋白Cx43,经计算机图像分析表明EGF使连接蛋白Cx43表达减弱,下调的程度与EGF的浓度呈正相关。LY染料传输试验显示在EGF作用下视网膜色素上皮细胞间通讯功能明显降低。结论体外培养的人RPE细胞表达Cx43,EGF可以减弱细胞间隙连接通讯功能、下调Cx43在人RPE细胞中的表达。     

表皮生长因子对人视网膜色素上皮细胞Cx43mRNA表达的影响

目的:观察表皮生长因子(epidermalgrowthfactor,EGF)对培养的人视网膜色素上皮(retinapigmentepitheliumcell,RPE)细胞中缝隙连接蛋白Cx43蛋白及mRNA的表达影响。方法:不同浓度的EGF作用于培养的第4代人的RPE细胞24h,采用Westernblot的方法测定Cx43的蛋白;原位杂交观察Cx43mRNA在RPE细胞中的表达特点;RT-PCR实验对EGF影响后的RPE细胞的mRNA进行半定量观察。结果:Westernblot试验证实不同浓度的EGF均可使Cx43蛋白表达量明显减少,并且与EGF的浓度呈正相关,原位杂交显示所有细胞均表达Cx43mRNA,其表达部位主要在细胞质和核;RT-PCR实验对mRNA进行半定量观察显示表皮生长因子使RPE细胞的mRNA表达量明显减少。结论:EGF可以下调Cx43蛋白及Cx43mRNA在人RPE细胞中的表达。     

氯化锂对人眼Tenon囊成纤维细胞TGF-β及CTGF的影响

目的:研究氯化锂(lithium chloride,LiCl)对体外培养的人眼Tenon囊成纤维细胞(human Tenon's capsule fibroblasts,HTFs)转化生长因子β(transforming growth factor-β,TGF-β)及结缔组织生长因子(connective tissue growth factor,CTGF)表达的影响并探讨其作用机制.方法:体外培养HTFs并通过vimentin免疫荧光染色技术及细胞形态特征观察鉴定细胞;将HTFs分为实验组(80mmol/L LiCl处理48h)和对照组(未用药).用实时荧光定量PCR(Real-time fluorescent quantitative polymerase chain reaction, Real time-qPCR)检测两组细胞内TGF-β及CTGF基因的表达,Western blot检测两组细胞内TGF-β及CTGF蛋白的表达.结果:体外培养的HTFs能够表达TGF-β及CTGF.与对照组比较,实验组中TGF-β、CTGF基因表达下降,差异均具有统计学意义(t=20.042、14.995,P<0.05);与对照组比较,实验组中TGF-β、CTGF蛋白表达下降,差异均具有统计学意义(t=46.058、12.452, P<0.05).结论:体外培养的HTFs在基因和蛋白水平表达TGF-β及CTGF,LiCl能够抑制该过程,其可能通过该机制抑制HTFs增殖,此研究为LiCl用于青光眼滤过术后瘢痕化调控提供了实验依据.     

阿昔洛韦对体外培养人眼Tenon囊成纤维细胞增殖迁移的影响

目的:探索阿昔洛韦(ACV)对体外培养人眼Tenon囊成纤维细胞(HTFs)的增殖和凋亡的影响以及作用机制。方法:将HTFs分为ACV处理组和空白组;CCK8检测不同浓度梯度下的细胞增殖速率,划痕实验检测HTFs迁移能力,流式细胞术检测HTFs凋亡以及细胞周期。结果:与空白组相比,ACV处理组(终浓度分别为1.125、2.25、3.375、4.5mmol/L)HTFs增殖速率显著降低(P<0.05),且呈浓度依赖性。4.5mmol/L ACV处理组划痕细胞迁移率显著降低(P<0.05),细胞凋亡率显著增加(P=0.0005),细胞周期G0/G1期峰值升高(P=0.0011),S期下降(P=0.0006),细胞周期被阻滞在G0/G1期。结论:阿昔洛韦可以通过阻滞HTFs周期促进细胞凋亡,抑制HTFs的增殖和迁移。     

维甲酸对人RPE细胞胞间通讯功能和Cx43表达的影响

目的观察培养的人视网膜色素上皮（RPE）细胞中连接蛋白Cx43的表达特点，以及维甲酸（RA）对人RPE细胞生长和细胞间缝隙连接功能、细胞间通道蛋白Cx43表达的影响及其相互关系。方法免疫组织化学方法观察缝隙连接蛋白（Cx43）在培养的人RPE细胞中的表达特点；不同浓度的RA作用于培养的第4代人的色素上皮细胞，用MTT方法观察RA对体外培养人RPE细胞生长的抑制；用LY染料传输方法测定细胞间隙连接功能，观察这三组不同浓度的RA对人RPE细胞缝隙连接功能的影响；免疫组织化学方法鉴别不同浓度RA影响后的缝隙连接蛋白Cx43的表达，用计算机灰度测量的方法评估表达的强度并作统计学分析。结果免疫组织化学染色显示体外培养的人RPE细胞表达缝隙连接蛋白Cx43，表达的部位主要在细胞浆和细胞膜上；RA对人RPE细胞的生长有明显的抑制作用，其抑制作用呈剂量依赖性；LY染料传输试验表明在RA的作用下RPE细胞间通讯功能明显增强；经计算机图像分析表明RA使连接蛋白Cx43表达增强，增强的程度与RA的浓度呈正相关。结论体外培养的人RPE细胞可以表达Cx43蛋白，RA抑制培养的色素上皮细胞的生长，提高细胞间隙连接通讯功能，上调Cx43蛋白在人RPE细胞中的表达。     

羟基喜树碱对人眼Tenon蒺s囊成纤维细胞的抑制作用

目的：研究羟基喜树碱（HCPT）对人眼Tenon＇s囊成纤维细胞（human Tenon＇s capsule fibroblasts,HTFs）的细胞周期、细胞毒性的影响,探讨其抗纤维化作用机制。方法：取本院眼库新鲜眼球（〈6h）的Tenon＇s囊组织,用组织块培养法进行成纤维细胞体外培养;流式细胞仪测定HCPT和丝裂霉素C（MMC）对HTFs细胞周期的影响;台盼蓝染色法鉴定HCPT和MMC对HTFs的抑制作用是否由药物的细胞毒性引起;RT-PCR检测HCPT、MMC作用24h后HTFs的Smad7 mRNA基因表达。结果：HTFs体外生长良好,可用于实验研究。不同浓度HCPT（0,0.25,1,4mg/L）作用后的HTFs细胞周期与对照组相比,G2期细胞百分数的组间差异均有统计学意义（P〈0.05）;不同浓度MMC（0,0.025,0.1,0.4mg/L）作用后的HTFs细胞周期与对照组相比,G1期细胞百分数的组间差异均有统计学意义（P〈0.05）。不同浓度HCPT和MMC作用后的HTFs活细胞率和空白对照组比较,差异均无统计学意义（P〉0.05）。4mg/LHCPT,0.4mg/LMMC作用HTFs24h后,Smad7 mRNA表达水平与空白对照组比较差异有统计学意义（P〈0.05）。结论：HCPT将HTFs阻滞于G2期,MMC将HTFs阻滞于G1期;HCPT,MMC抑制HTFs的作用和药物的细胞毒性无关;HCPT抑制HTFs的增殖可能是通过上调Smad7mRNA的表达阻断TGF-茁信号通路实现的。     

The protective effect of functional connexin43 channels on a human epithelial cell line exposed to oxidative stress

PURPOSE: To determine the role of connexin43 (Cx43) and gap junctional intercellular communication (GJIC) in the response of the human retinal pigment epithelial cell line ARPE-19 to oxidative stress. METHODS: ARPE-19 cells were treated with the chemical oxidant tert-butyl hydroperoxide (t-BOOH), and cell viability was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. GJIC was evaluated by scrape loading/dye transfer and microinjection assays, and Cx43 expression was detected by Western blot and immunofluorescent staining combined with confocal microscopy analysis. Retroviral infection of ARPE-19 cells with shRNA vectors targeting Cx43 or vectors encoding Cx43, Cx26, and a disease-linked dominant negative Cx43 mutant (G21R) were used, and the effect on cell viability was assessed. RESULTS: t-BOOH-induced ARPE-19 cell death was correlated with reductions in GJIC and in the total level of Cx43 protein expression. Overexpression of Cx26 and Cx43 increased the viability of oxidant-treated ARPE-19 cells. Conversely, shRNA knockdown of Cx43, expression of a disease-linked dominant negative Cx43 mutant, and blocking GJIC with 18beta-glycyrrhetinic acid and flufenamic acid all increased t-BOOH-induced ARPE-19 cell death. CONCLUSIONS: Cx43-mediated protection of ARPE-19 cells from oxidative stress-induced death is dependent on functional Cx43 channels.     

High glucose alters connexin 43 expression and gap junction intercellular communication activity in retinal pericytes

PURPOSE: To investigate the role of the gap junction protein, connexin-43 (Cx43) in the maintenance of retinal vascular homeostasis in diabetic retinopathy. METHODS: In human retinal pericytes (HRPs) and bovine retinal pericytes (BRPs) grown for 7 days in normal (5 mM) or high (30 mM)-glucose medium, the Cx43 protein level was determined by Western blot analysis. Parallel experiments were performed in HRPs to determine the Cx43 mRNA level by RT-PCR, the distribution and localization of Cx43 protein by immunostaining, and gap junction intercellular communication (GJIC) activity by a scrape-loading dye transfer technique. Distribution and localization of Cx43 protein was also determined in pericyte-endothelial cell cocultures. RESULTS: Western blot analysis of the Cx43 protein level in HRPs and BRPs indicated reduced Cx43 expression in the high-glucose condition (69.1% +/- 17% of control, P = 0.004; 62.3% +/- 19% of control, P = 0.001, respectively). The Cx43 mRNA level in HRPs grown in high-glucose medium also showed significant reduction (71.4% +/- 16.8% of control, P = 0.02). The relative number of Cx43 plaques indicative of Cx43 localization at specific sites of contact between adjacent cells showed significant reduction in the high-glucose condition (61% +/- 10% of control, P = 0.002); similarly, a significant reduction in the number of plaques was observed in cocultures grown in high-glucose medium compared with those in normal medium (59.4% +/- 29% of control, P = 0.001). Cells with reduced Cx43 expression showed significantly reduced transfer of lucifer yellow (61% +/- 13% of control, P = 0.001; r = 0.9). CONCLUSIONS: High-glucose-induced downregulation of Cx43 expression and inhibition of GJIC in retinal pericytes may play a role in the disruption of vascular homeostasis in diabetic retinopathy.     

Connexin 43 expression and proliferation of human limbal epithelium on intact and denuded amniotic membrane.

PURPOSE: Stem cell (SC)-containing limbal basal epithelium and transient amplifying cell (TAC)-containing corneal basal epithelium lie on different mesenchymal matrices. The gap junction protein connexin 43 (Cx43) is absent in the limbal basal epithelium but is present in the corneal basal epithelium, suggesting that the expression of Cx43 denotes SC differentiation into TACs. Amniotic membrane (AM) can expand limbal epithelial progenitor cells in vivo and in culture for subsequent corneal surface reconstruction. In this study, the modulation of Cx43 expression, gap junction intercellular communication (GJIC), and proliferative activity of ex vivo expanded human limbal epithelial (HLE) cells on intact and epithelially denuded AM was investigated. METHODS: HLE cells were expanded on intact (i.e., remaining devitalized amniotic epithelium) or epithelially denuded AM (EDTA-treated). Cx43 expression and 24-hour 5-bromo-2'-deoxyuridine-5'monophosphate (BrdU) labeling index (percentage) were determined by double immunostaining. GJIC was investigated by a scrape-loading dye transfer assay. In a subset of cultures Cx43 and K3 keratin as well as BrdU-retaining nuclei were analyzed in the stratified epithelium obtained 5 days after subcutaneous transplantation in NIH bg-nu-xidBR mice of AM cultures continuously labeled with BrdU for 7 days. RESULTS: The outgrowth rate, overall, was significantly higher on EDTA-treated AM than on intact AM (P < 0.05). Cx43 was expressed in 12.4% +/- 14.5% (n = 5) on intact and 57.5% +/- 18.2% (n = 5) on EDTA-treated AM (P < 0.05). The BrdU labeling index was 2.4% +/- 0.9% (n = 5) for the intact AM group, which was significantly less than 22.5% +/- 8.2% (n = 5) for EDTA-treated AM (P < 0.05). BrdU-labeled cells did not express Cx43. The dye transfer assay revealed reduced GJIC on both AM-cultured groups compared with the control culture on plastic (P < 0.002). GJIC on intact AM (17%) was reduced compared with that on EDTA-treated AM (27%; P = 0.42). After xenotransplantation, the basal layer of the stratified epithelium was Cx43 and K3 keratin negative and retained BrdU on intact AM, resembling characteristics of the limbal basal epithelium in vivo. In contrast, that of EDTA-treated AM was Cx43 and K3 keratin positive without BrdU retention, resembling characteristics of the corneal epithelium in vivo. CONCLUSION: These data indicate that denudation of the devitalized amniotic epithelium to expose its basement membrane might be a microenvironmental cue to promote TAC differentiation. The model system described herein is ideal for future exploration of the exact mechanistic operation in the microenvironmental niche that maintains the "stemness" of limbal SCs as well as in the signal that promotes corneal TAC differentiation.     

Regulation of gap junction intercellular communication in primary canine lens epithelial cells: role of protein kinase C

PURPOSE: Gap junction intercellular communication (GJIC) is important in maintaining lens epithelial cell homeostasis and reductions in GJIC may be associated with the development of cataract. Protein kinase C (PKC) activation can disrupt gap junction communication via phosphorylation of connexin 43 (C x 43) proteins that compose gap junction channels. This study examined the role of PKC activation in modulating GJIC in a primary canine lens epithelial cell (LEC) line. METHODS: TPA (12-O-tetradecanoyl-phorbol-acetate), a potent PKC activator and inhibitor of GJIC, was utilized in the present study. Primary cultures of canine LEC were treated with TPA (0-1000 ng/ml) for 0.5 hr and GJIC was assessed by scrape loading/dye transfer (SL/DT), and immunoblotting to detect phosphorylation of C x 43 protein. Inhibition of general and calcium-dependent PKC activity was achieved by pretreatment of cells with GF109203X and G?6976, respectively. RESULTS: Treatment with TPA (1-1000 ng/ml) significantly decreased GJIC in canine LEC as assessed by SL/DT. Pretreatment with 10 and 100 ng/ml TPA decreased GJIC by 80% as compared to controls and increased Cx43 phosphorylation as assessed by immunoblotting. Pretreatment of cells with GF109203X and G?6976, partially restored TPA-inhibited GJIC by 40% and 60%, respectively, and reduced C x 43 phosphorylation. Expression of calcium dependent PKC isoforms was detected in canine whole lens and LEC. CONCLUSIONS: Treatment with TPA significantly reduces GJIC in canine LEC. These effects are mediated, in part, by activation of calcium-dependent PKC isoforms. Primary canine LEC are a useful model in the study of the molecular mechanisms involved in GJIC and cataractogenesis.     

Upregulation and maintenance of gap junctional communication in lens cells

The cells of the lens are joined by an extensive network of gap junction intercellular channels consisting of connexins 43, 46, and 50. We have proposed, and experimentally supported, the hypothesis that fibroblast growth factor (FGF) signaling is required for upregulation of gap junction-mediated intercellular coupling (GJIC) at the lens equator. The ability of FGF to increase GJIC in cultured lens cells requires sustained activation of extracellular signal-regulated kinase (ERK). In other cell types, activation of ERK has been shown to block GJIC mediated by connexin43 (Cx43). Why ERK signaling does not block lens cell coupling is not known. Another unresolved issue in lens gap junction regulation is how connexins, synthesized before the loss of biosynthetic organelles in mature lens fiber cells, avoid degradation during formation of the organelle-free zone. We have addressed these questions using serum-free cultures (termed DCDMLs) of primary embryonic chick lens epithelial cells. We show that FGF stimulates ERK in DCDMLs via the canonical Ras/Raf1 pathway, and that the reason that neither basal nor growth factor-stimulated GJIC is blocked by activation of ERK is because it is not mediated by Cx43. In fibroblastic cells, the normally rapid rate of degradation of Cx43 after its transport to the plasma membrane is reduced by treatments that either directly (ALLN; epoxomicin) or indirectly (generation of oxidatively un/mis-folded proteins by arsenic compounds) prevent the ubiquitin/proteasome system (UPS) from acting on its normal substrates. We show here that Cx45.6 and Cx56, the chick orthologs of mammalian Cx50 and Cx46, behave similarly in DCDMLs. When organelles lyse during the maturation of fiber cells, they release into the cytosol a large amount of new proteins that have the potential to saturate the capacity, and/or compromise the function, of the UPS. This would serve to spare gap junctions from degradation during formation of the organelle-free zone, thereby preserving GJIC between mature fiber cells despite the lack of de novo connexin synthesis.     

The effect of connexin43 on the level of vascular endothelial growth factor in human retinal pigment epithelial cells

Background  

Connexins (Cx) are the basic units of gap junctions and contribute to cellular integrity by promoting intercellular communication. Disruption of the retinal pigment epithelial monolayer may be an early event in the pathogenesis of age-related macular degeneration, a condition in which vascular endothelial growth factor (VEGF) is known to be of importance. This study was designed to assess the effect of connexin43 (Cx43) expression and gap junctional intercellular communication (GJIC) on the expression and secretion of VEGF from the retinal pigment epithelium under normal cell culture and oxidative stress conditions.     

Changes in connexin43 in early ocular surface development

PURPOSE: In the limbo-corneal epithelium the stem and early precursor epithelial cell pool is confined to the limbal rim. Among the features associated with this spatial segregation is the general paucity of connexin43 (Cx43) within the limbal basal cell population and its complete absence in resident stem cells. The limbo-corneal epithelial lineage derives from a Cx43-positive (Cx43+) embryonic outer ectoderm. Accordingly, as a means of identifying the process through which limbal cell phenotypes emerge, we investigated the expression of Cx43 in the ocular surface of embryonic rats. METHODS: Ocular surface expression of Cx43 or K12 was determined in cryostat sections of rat embryos and eyes using immunohistological methods. RESULTS: Changes in Cx43 expression revealed the early phenotypic divergence of three main epithelial cell phenotypes of the ocular surface. An analysis of the level and distribution pattern of Cx43 puncta lead to the identification of two distinct domains by embryonic day 10 (E10), a stage that occurs soon after formation of the lens vesicle. Additionally, at E12, ectodermal cells directly adjacent to the edges of the developing retina no longer express connexin. A comparison of anatomical and expression changes throughout embryonic development demonstrated that the two early zones represent the rudiments for the epithelia of the central cornea and conjunctiva, respectively, and that the isolated Cx43-negative (Cx43-) cells represent the precursors of the basal and, putatively, stem cells of the limbal epithelium. CONCLUSIONS: Changes in Cx43 expression revealed that the phenotypic divergence of ocular surface epithelial cells and the generation of limbo-corneal stem cell precursors takes place at a very early stage in ocular development, ahead of the establishment of any identifiable anatomical or differentiation features for these domains.     

Gap junctional intercellular communication in bovine corneal endothelial cells

Gap junctions and/or paracrine mediators, such as ATP, mediate intercellular communication (IC) in non-excitable cells. This study investigates the contribution of gap junctions toward IC during propagation of Ca(2+) waves in cultured bovine corneal endothelial cells (BCEC) elicited by applying a point mechanical stimulus to a single cell in a confluent monolayer. Changes in [Ca(2+)](i) were visualized using the fluorescent dye Fluo-4. The area reached by the Ca(2+) wave, called the active area (AA), was determined as a measure of efficacy of IC. RT-PCR and Western blotting showed expression of Cx43, a major form of connexin, in BCEC. In scrape-loading (using lucifer yellow) and fluorescence recovery after photobleaching (FRAP; using carboxyfluorescein) protocols, significant dye transfer of the hydrophilic dyes was evident indicating functional gap junctional IC (GJIC) in BCEC. Gap27 (300 microM), a connexin mimetic peptide that blocks gap junctions formed by Cx43, reduced the fluorescence recovery in FRAP experiments by 19%. Gap27 also reduced the active area of the Ca(2+) wave induced by point mechanical stimulation from 73,689 microm(2) to 26,936 microm(2), implying that GJIC contribution to the spread of the wave is at least approximately 63%. Inhibitors of ATP-mediated paracrine IC (PIC), such as a combination of apyrase VI and apyrase VII (5U/ml each; exogenous ATPases), suramin (200 microM; P2Y antagonist), or Gap26 (300 microM; blocker of Cx43 hemichannels) reduced the active area by 91%, 67%, and 55%, respectively. Therefore, estimating the contribution of GJIC from the residual active area after PIC inhibition appears to suggest that GJIC contributes no more than approximately 9% towards the active area of the Ca(2+) wave. Gap27 did not affect the enhancement in active area induced by ARL-67156 (200 microM, ectonucleotidase inhibitor), ATP release induced by point mechanical stimulation, and zero [Ca(2+)](o)-induced lucifer yellow uptake, indicating that the peptide has no influence on PIC. Exposure to Gap27 in the presence of PIC inhibitors led to a significant further inhibition of the Ca(2+) wave. The finding that the residual active area after inhibition of PIC by apyrases was much smaller than the reduction of the active area by Gap27, provides evidence for interaction between GJIC and PIC. These findings together suggest that functional gap junctions are present in BCEC, that both GJIC and PIC contribute significantly to IC, and that the two pathways interact.     

Protein kinase Cgamma regulation of gap junction activity through caveolin-1-containing lipid rafts

PURPOSE: To demonstrate the interactions of PKCgamma with caveolin (Cav)-1 and connexin(Cx)43 in lipid rafts and its regulation of gap junctions. METHODS: N/N1003A lens epithelial cells, bovine primary lens epithelial cells, and stably transfected N/N1003A lens epithelial cells were used. Coimmunoprecipitation and Western blot analysis were used to detect protein expression and their interactions. Cav-1-containing lipid rafts and redistribution of Cav-1, PKCgamma, and Cx43 were analyzed by sucrose gradients and by consequent Western blot analysis. Cell surface gap junction Cx43 plaques were detected by confocal microscopy. PKCgamma activity was measured with a PKC assay kit. RESULTS: Cav-1 and -2 were found in N/N1003A and bovine primary lens epithelial cells. Cx43 was associated with Cav-1 in lipid rafts. Phorbol ester (TPA) and insulin-like growth factor (IGF)-1 recruited PKCgamma into Cav-1-containing lipid rafts and stimulated the interactions of PKCgamma with Cav-1 and Cx43. TPA and IGF-1 induced redistribution of Cav-1 and Cx43 from light-density fractions to higher density fractions on sucrose gradients. PKCgamma redistributed with Cav-1- and Cx43-containing fractions on stimulation with TPA or IGF-1. Overexpression of PKCgamma-enhanced green fluorescent protein (EGFP) increased the interaction of PKCgamma-EGFP with Cav-1 and Cx43 and decreased gap junction Cx43 plaques without exogenous growth factors. Overexpression of a loss-of-function PKCgamma mutant did not decrease gap junction Cx43 plaques or cause redistribution in lipid rafts, even though the PKCgamma mutant still interacted with Cav-1 and Cx43. CONCLUSIONS: Activation of PKCgamma by TPA or IGF-1 stimulated the interaction of PKCgamma with Cav-1 and Cx43 in lipid rafts, causing Cx43, Cav-1, and PKCgamma to redistribute within the lipid rafts, and this resulted in a decrease in gap junction plaques.