不同浓度多西环素对碱烧伤大鼠角膜中转化生长因子β1和基质金属蛋白酶9表达的影响

目的　探讨不同浓度多西环素对碱烧伤大鼠角膜中转化生长因子β1（transforming growth factor-β1，TGF-β1）和基质金属蛋白酶-9（matrix metalloproteinase-9，MMP-9）表达的影响及其调节作用。方法　SD大鼠右眼制备成角膜碱烧伤模型，随机分为对照组、0.5 g·L^-1多西环素组和1.0 g·L^-1多西环素组，每组15只。3组分别于造模后每天给予20 μL眼液溶媒、0.5 g·L^-1多西环素、1.0 g·L^-1多西环素滴眼至观察期末。于碱烧伤后第3天、第7天、第14天对角膜混浊程度进行评分，并行RT-PCR和ELISA检测角膜组织中TGF-β1、MMP-9 mRNA和蛋白的表达情况。结果　与对照组相比，碱烧伤后第7天、第14天，0.5 g·L^-1多西环素组［（2.80±0.45）分、（2.20±0.45）分］和1.0 g·L^-1多西环素组［（2.00±0.00）分、（0.60±0.55）分］角膜混浊程度评分降低，且1.0 g·L^-1多西环素组更低，差异均有统计学意义（均为P＜0.05）。碱烧伤后第3天、第7天、第14 天，0.5 g·L^-1多西环素组、1.0 g·L^-1多西环素组TGF-β1 mRNA及蛋白表达量均较对照组低，且1.0 g·L^-1多西环素组表达更低，差异均有统计学意义（均为P＜0.05）。碱烧伤后第3天、第7天，0.5 g·L^-1多西环素组、1.0 g·L^-1多西环素组MMP-9 mRNA及蛋白表达均较对照组低，且1.0 g·L^-1多西环素组表达更低，差异均有统计学意义（均为P＜0.05）。结论　多西环素可下调碱烧伤大鼠角膜中TGF-β1和MMP-9的表达，促进角膜愈合，并呈一定程度的剂量依赖性。     

TGF-β对体外培养人胚视网膜色素上皮细胞的影响

目的 研究转化生长因子-β（TGF-β）对体外培养人胚视网膜色素上皮（RPE）细胞的影响，从细胞生物学水平和分子生物学水平探讨近视眼的发病机制。方法 外源性TGF-β刺激人RPE细胞后，通过绘制生长曲线、MTT法检测细胞活性及观察透射电镜研究其细胞自身的生长情况；采用酶联免疫吸附实验（ELISA）测定其分泌HGF、bFGF和TGF-β的量；采用RT-PCR法检测其细胞表达HGF的情况，从而分析外源性TGF-β对体外培养的RPE细胞的影响。结果不同质量浓度的TGF-β均可抑制人胚RPE细胞的生长，随时间的延长作用更加明显；TGF-β可使RPE细胞超微结构发生变化；ELISA结果显示TGF-β对其他两种因子的分泌均有抑制作用，但对自身有促进作用，为正反馈调节；同时可明显抑制RPE细胞内HGF的表达。结论 TGF-β可影响RPE细胞增生、改变超微结构及抑制HGF的表达。     

转化生长因子-β1对人视网膜色素上皮细胞热休克蛋白47及血管内皮生长因子表达的影响

人视网膜色素上皮(RPE)细胞增生、迁移、合成细胞外基质是促进增生性玻璃体视网膜疾病(PVD)发生和发展的主要原因之一[1-3].血管内皮生长因子(VEGF)可诱导入RPE细胞增生、迁移;转化生长因子-β1(TGF-β1)可诱导入RPE细胞迁移及合成细胞外基质;均在PVD形成过程中发挥着重要作用[2-4].但目前,有关其作用机制尚不明确.RPE细胞热休克蛋白47(HSP47)是一种胶原特异的分子伴侣,对胶原成熟有一定影响[5].我们通过观察TGF-β1对人RPE细胞HSP47及VEGF合成的影响,以进一步探讨TGF-β1在PVD发病机制中的作用.现将结果报道如下.     

TGF-β1对RPE细胞bcl-2/Fas mRNA和Caspase-3表达的影响

目的观察转化生长因子-β1（transforming grawthfactor-β1，TGF-β1）诱导人视网膜色素上皮细胞（retinal pigment epithelium，RPE）凋亡的作用机制。方法使用逆转录多聚酶链反应检测不同浓度TGF-β1作用下的人RPE细胞中凋亡相关基因bel-2和Fas mRNA的表达。使用Western-Blot蛋白印迹法检测不同浓度TGF-β1处理过的PRE细胞中Caspase-3蛋白的表达。结果逆转录多聚酶链反应正示，随着TGF-β1岛浓度的增高，人RPE细胞中bcl-2mRNA表达降低，Fas mRNA的表达逐渐增高；Western-Blot结果显示。随着TGF-β1浓度的增高，Caspase-3蛋白表达逐渐增高。结论TGF-β1可上调RPE细胞表面Fas基因的表达，从而激活Caspase-3的活性而诱导RPE细胞凋亡，Caspase-3在RPE细胞凋亡过程中起着非常重要的作用。[眼科新进展2006；26（3）：194-197]     

缓激肽对转化生长因子-β1（TGF-β1）诱导视网膜色素上皮细胞发生上皮间充质转化的影响

目的　制作增生性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR)的上皮间充质转化（epithelial-mesenchymal transformation,EMT）细胞模型,研究缓激肽（bradykinin,BK）对视网膜色素上皮（retinal pigment epithelium,RPE）细胞发生EMT的影响,并探讨BK对PVR的影响机制。方法　体外培养人RPE细胞株ARPE-19细胞,采用不同浓度转化生长因子-β1（transforming growth factor- β1,TGF- β1）分别作用于ARPE-19细胞24 h、48 h,于倒置显微镜下观察细胞形态变化;采用CCK-8检测细胞增殖情况,确定TGF-β1浓度及作用时间;利用Western blot和细胞免疫荧光检测EMT标志蛋白E-钙黏素、α-平滑肌肌动蛋白（α-smooth muscle actin,α-SMA）和波形蛋白（Viminten）表达情况;细胞划痕实验、Transwell实验检测细胞迁移能力。同时采用Western blot检测TGF/Smad通路下游pSmad3和Smad7的表达。结果　TGF- β1刺激ARPE-19细胞后可以成功诱导EMT体外细胞模型。当TGF-β1浓度为10 μg·L^-1、作用时间为48 h时,细胞增殖最明显。BK在TGF- β1诱导的EMT中可以降低α-SMA、Viminten的表达,升高E-钙黏素的表达并且降低细胞迁移能力。这些影响能被BK-2受体拮抗剂HOE-140逆转。TGF-β1诱导ARPE-19细胞发生EMT时,pSmad3表达量升高;TGF-β1刺激前给予BK刺激,pSmad3表达量减少;加入BK前预敷HOE-140,然后给予TGF-β1刺激,BK作用减弱,pSmad3表达量升高。Smad7表达趋势与pSmad3表达趋势相反。结论　10 μg·L^-1 TGF-β1可导致ARPE-19细胞发生EMT。BK通过TGF-/Smad信号通路上调Smad 7的表达、下调pSmad 3的表达,从而逆转TGF-β1诱导的EMT。提示BK可能是一种新的、有效的治疗PVR的方法。     

巨噬细胞条件培养液及重组人IL-1β对RPE细胞产生MMP-2及MMP-9的影响

目的 观察巨噬细胞条件培养液（MφM）及白细胞介素-1β（IL-1β）对视网膜色素上皮（RPE）细胞分泌基质金属蛋白酶（MMP）-2及MMP-9的影响,探讨巨噬细胞与RPE细胞之间的相互关系.方法 在人RPE细胞培养液中加入50%体积分数的人MφM作用48 h;加入终质量浓度为10、50、100、200 ng/ml重组人IL-1β作用12、24、48 h.用酶谱法检测RPE细胞培养液中MMP-2及MMP-9,凝胶图像扫描分析仪定量.结果 MφCM促RPE细胞分泌MMP-2和MMP-9,促MMP-9分泌尤其明显;48 h内IL-1β促RPE细胞分泌MMP-2和MMP-9具有时间和质量浓度依赖性.结论 MφCM中含有促RPE细胞分泌MMP-2和MMP-9的因子,IL-1β是巨噬细胞产生的主要炎性因子,提示巨噬细胞可能通过分泌细胞因子调节RPE细胞分泌MMP-2和MMP-9,参与细胞增生、迁移等病理过程.     

ALK5抑制剂对转化生长因子-β1（TGF-β1）致视网膜色素上皮细胞纤维化的干预作用

目的　探讨转化生长因子-β1（transforming growth factor-beta1，TGF-β1）对人视网膜色素上皮（retinal pigment epithelial，RPE）细胞向成纤维细胞转化的影响，及活化素受体样激酶5（activated receptor kinase 5，ALK5）抑制剂（SB-431542）对这一影响的干预作用。方法　体外培养人RPE细胞，随机分为对照组、TGF-β1处理组、TGF-β1+SB-431542处理组、SB-431542处理组。对照组不做任何处理，其余三组分别用10 μg·L^-1 TGF-β1、10 μg·L^-1 TGF-β1+30 μmol·L^-1 SB-431542、30 μmol·L^-1 SB-431542处理细胞24 h。MTT法检测各组RPE细胞增殖率；划痕实验观察各组处理后0 h、12 h、24 h RPE细胞迁移情况；免疫荧光法检测各组细胞ALK5蛋白的分布和表达；Western blot法检测各组ALK5、血管内皮生长因子（vascular endothelial growth factor，VEGF）、α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）、Ⅰ型胶原（collagen typeⅠ，ColⅠ）以及纤维粘连素（fibronectin，FN）蛋白的表达。结果　与对照组相比，TGF-β1作用于RPE细胞24 h后，可改变RPE细胞表型，使其呈成纤维细胞样外观，并明显促进RPE细胞增殖和迁移。10.0 g·L^-1 TGF-β1作用24 h后RPE细胞增殖率为对照组的（1.33±0.08）倍，30 μmol·L^-1 SB-431542作用24 h后RPE细胞增殖率为10.0 g·L^-1 TGF-β1处理组的（67.77±6.78）%。经TGF-β1作用24 h后RPE细胞内ALK5、VEGF、α-SMA、ColⅠ及FN蛋白的表达显著上调，分别是对照组的（3.69±0.37）倍、（1.76±0.05）倍、（2.58±0.18）倍、（1.86±0.11）倍、（1.74±0.08）倍；SB-431542可逆转TGF-β1诱导的ALK5、VEGF、α-SMA、ColⅠ及FN蛋白表达上调，分别是TGF-β1处理组的（67.73±5.15）%、（71.71±3.50）%、（79.87±0.05）%、（63.59±3.16）%、（83.07±2.31）%，差异均有统计学意义（均为P＜0.05）。结论　TGF-β1可促进RPE细胞增殖、迁移，向成纤维细胞转化，并能显著上调RPE细胞内ALK5、VEGF、α-SMA、ColⅠ及FN蛋白的表达；SB-431542可通过抑制TGF-β1的作用而抑制RPE细胞的纤维化。     

多西环素对培养的人结膜上皮细胞炎性反应及凋亡的影响

目的探讨多西环素对体外培养的人结膜上皮细胞细胞黏附分子1（ICAM-1,CD54）、人类白细胞抗原DR（HLA—DR）、白细胞介素1β（IL-1β）表达及凋亡的影响。方法混合消化液培养法培养人结膜上皮细胞,免疫组织化学方法进行细胞鉴定。培养至第3代或第4代,在细胞培养液中分别加入γ干扰素（IFN-γ）0U／ml、IFN-γ300U／ml、IFN-γ300U／ml＋多西环素10μg／ml、IFN-γ300U／ml＋多西环素20μg／ml、IFN-γ300U／ml＋多西环素40μg／ml、IFN-γ300U／ml＋地塞米松100μg／ml,24h后收集细胞,流式细胞学检测CD54、HLA—DR及IL-1β的表达,Western blot检测CD54的表达。72h后收集细胞,碘化丙啶（PI）染色,流式细胞学检测凋亡。结果培养的正常人结膜上皮细胞可以表达少许CD54和IL-1β,加入IFN-γ后表达明显增加,加入多西环素和地塞米松后CD54和IL-1β的表达下降（P〈0．01）。随着所加多西环素浓度的增加,CD54和IL-1β的表达逐渐降低（P〈0．01）。培养的正常人结膜上皮细胞未发现明显的HLA—DR的表达及凋亡的产生,加入IFN-γ及多西环素后HLA—DR的表达及凋亡水平无明显变化（P〉0．05）。结论多西环素可抑制培养的正常人结膜上皮细胞CD54和IL-1β等炎性相关因子的产生,提示多西环素对干眼等非感染性眼表炎性疾病的治疗可能具有一定的应用前景。（中华眼科杂志,2005,41：842-846）     

PDGF对人RPE细胞中MMP-2、MMP-9和TIMP-1表达的影响

背景研究表明血小板源性生长因子（PDGF）能调节多种细胞中基质金属蛋白酶／基质金属蛋白酶组织抑制剂（MMP／TIMP）的表达和平衡,但视网膜色素上皮（RPE）细胞中MMP／TIMP的表达与PDGF作用剂量和作用时间的关系尚不明确。目的观察PDGF对RPE细胞中MIP-2、MMP-9和TIMP-1表达的影响。方法体外培养人RPE细胞系ARPE-19,将达到70％～80％融合的细胞分为5个组。分别将0、0．1、1、10、50mg／LPDGF加入RPE细胞培养基作用36h,分别采用逆转录PCR（RT—RCR）法和Western blot法检测各组RPE细胞中MMP-2、MMP-9和TIMP-1 mRNA及其蛋白的表达。PDGF组采用10mg／LPDGF组PDGF分别刺激RPE细胞24、36和48h,对照组用不含PDGF的培养液培养,采用逆转录PCR（RT—RCR法和Western blot法分别检测RPE细胞中MMP-2、MMP-9和TIMP-1 mRNA及蛋白的表达。结果随着PDGF质量浓度的增加和刺激时间的延长,RPE细胞生长速度加快,细胞增生明显。PDGF刺激RPE细胞36h,随着PDGF质量浓度的增加,MMP-2 mRNA和MMP-9 mRNA在RPE细胞中表达相对值逐渐增加,各组间差异均有统计学意义（MMP-2 mRNA：F=79．304,P=0．000;MMP-9 mRNA：F=8．465,P=0．003）,其中1、10、50mg／LPDGF组RPE细胞中MMP-2 mRNA和MMP-9 mRNA表达相对值明显高于0mg／L PDGF组,差异均有统计学意义（P〈0．05）。RPE细胞中MMP-2和MMP-9蛋白表达相对值随着PDGF质量浓度的增加而逐渐增加,各组间差异均有统计学意义（MMP-2：F=26．550,P=0．000;MMP-9：F=80．993,P=0．000）,其中1、10、50mg／LPDGF组RPE细胞中MMP-2和MMP-9蛋白表达相对值明显高于0mg／L PDGF组,差异均有统计学意义（P〈0．05）。各质量浓度PDGF组RPE细胞中TIMP-1 mRNA和蛋白表达相对值差异均无统计学意义（F=0．143,P=0．962;F=1．955,P=0．178）。随着10mg／L PDGF刺激RPE细胞时间的延长,RPE细胞中MMP-2 mRNA和MMP-9 mRNA表达逐渐增加,差异均有统计学意义（MMP-2 mRNA：F时间=83．250,P=0．002;MMP-9 mRNA：F时间=6．785,P=0．019）;各时间点RPE细胞中MMP-2和MMP-9蛋白表达相对值明显增加,差异均有统计学意义（MMP-2：F时间=11．185,P=0．041;MMP-9：F时间=968．413,P=0．000）。PDGF作用不同时间点对照组与PDGF组间MMP-2、MMP-9 mRNA及蛋白在RPE细胞中的表达差异均有统计学意义（分组：均P=0．000,时间点：P〈0．05）,而各时间点2个组间RPE细胞中TIMP-1表达相对值的差异均无统计学意义（P〉0．05）。结论PDGF上调PRE细胞中MMP-2和MMP-9的表达,其作用呈剂量和时间依赖性,但对PRE细胞中TIMP-1的表达无明显影响。PDGF导致RPE细胞中MMP／TIMP的平衡失调,从而导致细胞外基质的破坏,促进RPE细胞的迁移。     

多西环素抑制视网膜母细胞瘤血管生成拟态的体外研究及组织学观察

研究多西环素对人视网膜母细胞瘤(RB)细胞体外血管生成拟态 （VM）和细胞增生的抑制效应。方法建立人RB细胞株三维培养系统,缺氧诱导后观察RB细胞能否形成血管网状结构及其特点;用5～20 mg/L不同浓度多西环素处理RB细胞, 过碘酸雪夫氏(PAS)染色观察对RB细胞体外血管生成拟态的抑制效应;噻唑蓝(MTT)比色法检测多西环素对RB细胞增生活性的变化; 逆转录聚合酶链式反应（RT-PCR）技术检测不同条件下基质金属蛋白酶2(MMP-2)、基质金属蛋白酶9(MMP-9)mRNA的相对表达量; 收集12例RB石蜡包埋样本进行CD34/PAS免疫组织化学双重染色,观察RB中的血管生成拟态。结果 RB细胞在三维培养系统形成管网状的拟态血管; 5～20 mg/L多西环素能抑制RB细胞体外管状结构数量。MTT检测显示多西环素加药组3、5、7 d的吸光度A［旧称光密度（OD）］值与对照组比较差异均有统计学意义(t=15.320,P <0.01),且不同浓度的多西环素组之间细胞的增生抑制率差异亦有统计学意义(F=9.442,11.729,12.120;P <0.05),其浓度与细胞的增生呈负相关(r=-0.924, P <0.01), 氯化钴缺氧组MMP-2、MMP-9的mRNA表达量明显增强,各浓度多西环素加药组显著低于氯化钴缺氧组(t=16.469,P <0.01)。在12例RB组织标本中可见CD34阴性PAS阳性的瘤细胞围成管道样结构,部分管腔中可见红细胞。结论 多西环素能够抑制RB体外血管生成拟态形成和细胞的增生,其机制可能是通过抑制MMP-2、MMP-9 mRNA的表达而实现。     

Modulation of matrix metalloproteinase and TIMP-1 expression by cytokines in human RPE cells

PURPOSE: The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) is crucial for homeostasis of ocular extracellular matrices. To assess altered MMP activity as a determinant in the migration of human retinal pigment epithelial (RPE) cells, expression characteristics of several MMPs and TIMP-1 in RPE cell cultures were investigated. METHODS: Expression studies were performed with RT-PCR, ELISA, and immunofluorescence analysis. Secretion of MMP-2 was demonstrated by zymography. Migration of cytokine-stimulated RPE cells was evaluated with microporous membranes of permeable chambers. RESULTS: MMP-1, -2, -3, and -9; MT2-MMP; and TIMP-1 were expressed in cultured RPE cells. MMP-2 was detected on the cell surface and in secreted inactive and active forms. TGF-beta(2), IL-1beta, and TNF-alpha enhanced secretion of MMP-1, -2, and -3. TGF-beta(2) also stimulated MT2-MMP cell surface expression and release of TIMP-1. The mRNA levels of MMP-1, -2, and -3 and TIMP-1 were markedly increased by TNF-alpha and TGF-beta(2). MMP-2 mRNA levels were also upregulated by PDGF-BB. Migration of RPE cells stimulated by TGF-beta(2) or PDGF-BB was inhibited in presence of a synthetic MMP inhibitor. CONCLUSIONS: Proinflammatory cytokines and TGF-beta(2) play an important role in the upregulation of expression of MMP-1, -2, and -3 in RPE cells and account for a directional shift in the balance between MMPs and TIMPs. Facilitation of RPE cell migration stimulated by cytokines (i.e., TGF-beta(2) or PDGF-BB) in ocular diseases may be due to increased release of MMPs, in the presence of comparatively lower levels of their inhibitors.     

The role of gremlin, a BMP antagonist, and epithelial-to-mesenchymal transition in proliferative vitreoretinopathy

PURPOSE: Proliferative vitreoretinopathy (PVR), a major reason for failure of retinal detachment surgery, is characterized by the formation of scarlike tissue that contains transdifferentiated retinal pigment epithelial (RPE) cells. The scar tissue occurs in response to growth factors such as transforming growth factor (TGF)-beta and epidermal growth factor (EGF). The authors postulate that transdifferentiation of RPE cells may arise via epithelial-to-mesenchymal transition (EMT). Bone morphogenetic proteins (BMPs) are expressed in the retina and have an antiproliferative role. Gremlin is expressed in the outer retina and is a BMP antagonist. The study was conducted to establish a model of PVR by inducing EMT in the human RPE cell line ARPE-19, using TGF-beta and EGF and to establish the contribution of gremlin to EMT. METHODS: ARPE-19 cells were cultured and stimulated with TGF-beta1, EGF, and gremlin. The expression of alpha-smooth muscle actin (alpha-SMA), vimentin, and zona occludens (ZO)-1 were examined via PCR, Western blot analysis, and immunofluorescence. Zymography was performed for matrix metalloproteinase (MMP) activity. Scratch assays were performed to assess migration. RESULTS: A model of EMT was established in the ARPE-19 cell line. The characteristics of EMT include gain of alpha-SMA, loss of ZO-1, upregulation of MMP activity and enhanced migration. Gremlin plays an important role in this process, contributing to the gain of alpha-SMA, loss of ZO-1, and upregulation of MMP activity. CONCLUSIONS: EMT occurs in vitro in the ARPE-19 cell line in response to the growth factors TGF-beta1 and EGF. EMT is also induced by Gremlin.     

肝细胞生长因子对培养的视网膜色素上皮 细胞移行及增生的作用

目的研究肝细胞生长因子(hepatocyte growth factor, HGF)对视网膜色素上皮 (retinal pigment epitheliaum, RPE) 细胞的促增生和促移行作用。方法在无血清培养液培养的人RPE细胞中分别加入1、2、10、50、100 μg/L不同浓度的HGF,四唑盐比色法(methyl thiazolyl tetrazolium, MTT)测定促细胞增生情况; 采用RPE细胞损伤愈合模型,在无血清培养液中分别加入1、2、10、50、100 μg/L 不同浓度的HGF,培养20 h后计数进入刮痕区的细胞,观察HGF对RPE细胞移行的作用。结果HGF浓度处于10 ～100 μg/L时对RPE细胞具有促增生作用(P<0.05或P<0.01), 增生率为18.2 %～34.8 %,其中浓度为50 μg/L作 用3 d达到最大促增生效果(P<0.01）,增生率为32.8 %;HGF可明显促进RPE细胞移行,促移行能力分别为113.0 %(10 μg/L), 91.7 %(50 μg/L) 和50.3 %(100 μg/ L )。浓度自2 μg/L开始出现促细胞移行作用(P<0.05),促移行能力为9.3 %。结论HGF可促进RPE细胞增生和移行,是RPE细胞的有丝分裂原和强力的促移行因子。(中华眼底病杂志,2001,17:307-310)     

Thrombin-induced VEGF expression in human retinal pigment epithelial cells

PURPOSE: The purpose of the present study was to investigate the effects of thrombin and thrombin in combination with other proangiogenic factors on VEGF expression in hRPE cells. METHODS: hRPE cells were stimulated with thrombin TNF-alpha, monocytes, and TGF-beta2. After stimulation, conditioned medium and lysed cells were subjected to ELISA, Western blot analysis, immunocytochemistry, and RT-PCR analyses. Inhibitors specific for various signal transduction pathways were used to determine the signaling pathways involved. RESULTS: Treatment of RPE cells with thrombin resulted in dose- and time-dependent increases in VEGF mRNA levels and protein production. hRPE VEGF expression is predominantly protease-activated receptor (PAR)-1 dependent. Approximately 80% of thrombin-induced VEGF secretion was abrogated by inhibitors of MAPK/ERK kinase (MEK), p38, c-Jun NH2-terminal kinase (JNK), protein tyrosine kinase (PTK), phosphatidylinositol 3-kinase (PI3K), protein kinase C (PKC), nuclear factor-kappaB (NF-kappaB), and reactive oxygen species (ROS). Analyses of VEGF protein production and mRNA synthesis revealed that VEGF induction by thrombin plus TNF-alpha or coculture with monocytes was additive, whereas that by co-incubation with TGF-beta2 was synergistic. The costimulated VEGF production by TGF-beta2 plus thrombin was an average of three times higher than the sum of that induced by each agent alone. Furthermore, BAPTA [bis-(o-aminophenoxy)ethane-N,N',N'-tetraacetic acid], a calcium chelator, blocked the VEGF secretion induced by thrombin and thrombin plus TGF-beta2 by 65% and 20%, respectively, but had no effect on that induced by TGF-beta2 alone. CONCLUSIONS: Thrombin alone and in combination with TNF-alpha, monocytes, and TGF-beta2 potently stimulated VEGF expression in hRPE cells via multiple signaling pathways. The thrombin-induced calcium mobilization may play an important permissive role in maximizing TGF-beta2-induced VEGF expression in RPE cells.     

Cell-Associated Human Retinal Pigment Epithelium Interleukin-8 and Monocyte Chemotactic Protein-1: Immunochemical and In-situ Hybridization Analyses

Human retinal pigment epithelial (RPE) cells secrete chemokines, interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) in response to pro-inflammatory cytokines. In this study we (1) examined the efficiency of human RPE IL-8 and MCP-1 secretion, (2) determined the amount of neutrophil and monocyte chemotactic activity in human RPE cell conditioned media and cell extracts that is attributable to IL-8 and MCP-1, respectively, and (3) assessed the sensitivity of immunohistochemistry and in situ hybridization for detecting chemokine production by cytokine-stimulated human RPE cells. Conditioned media and extracts from human RPE cells stimulated with various physiologic concentrations of interleukin-1 beta (IL-1β) (0.2–20 ng ml⁻¹), tumor necrosis factor (TNF-α) (0.2–20 ng ml⁻¹) or interferon-gamma (IFN-γ) (10–1000 U ml⁻¹) were examined to compare secreted and cell associated levels of IL-8 and MCP-1 at various time points up to 24 hr.ELISA demonstrated that IL-8 and MCP-1 are both efficiently secreted by pro-inflammatory cytokine treated human RPE cells. Substantial dose- and time-dependent RPE secretion of IL-8 was observed following stimulation with IL-1β or TNF-α, but cell associated IL-8 was detectable only after high dose (20 ng ml⁻¹) IL-1β stimulation and comprised less than 1% of the total IL-8 induced. Dose- and time-dependent RPE cell MCP-1 secretion was also observed following IL-1β>TNF-α>IFN-γ stimulation, with an average of 4% of the total MCP-1 retained within RPE. Bioassays demonstrated neutrophil and monocyte chemotactic activity in conditioned media from stimulated RPE cells, but not in human RPE cell extracts. Inhibition of conditioned media-induced chemotaxis by specific anti-IL-8 or anti-MCP-1 antibodies demonstrated that IL-8 and MCP-1 were responsible for the majority of HRPE-derived neutrophil (>60%) and monocyte (53–57%) chemotactic activity, respectively.Using in situ hybridization IL-8 mRNA was readily detected within IL-1β>TNF-α stimulated RPE cells and MCP-1 mRNA easily visualized within IL-1β>TNF-α> or IFN-γstimulated cells. Immunohistochemistry to detect IL-8 was positive only in RPE cells exposed to high dose IL-1β(20 ng ml⁻¹) for 8 or 24 hr and was weak. Immunohistochemical staining for MCP-1 in RPE cells was more intense and was visualized within RPE cells stimulated with IL-β, TNF-α, or IFN-γ. This study demonstrates that: (1) RPE cells efficiently secrete IL-8 and MCP-1 upon stimulation with pro-inflammatory cytokines; (2) secreted IL-8 and MCP-1 account for the majority of human RPE neutrophil and monocyte chemotactic activity; (3) in situ hybridization readily detects IL-8 and MCP-1 mRNA in cytokine stimulated RPE cells; and (4) immunohistochemistry demonstrates cell-associated MCP-1 in cytokine stimulated RPE cells, but only minimal cell-associated IL-8.     

Effect of cyclical mechanical stretch and exogenous transforming growth factor-beta1 on matrix metalloproteinase-2 activity in lamina cribrosa cells from the human optic nerve head

PURPOSE: Extensive remodeling of the lamina cribrosa extracellular matrix occurs in primary open angle glaucoma. The transforming growth factor-beta (TGF-beta) and matrix metalloproteinase (MMP) protein families are implicated in this process. The authors investigated (a). the effect of cyclical mechanical stretch on TGF-beta1 mRNA synthesis, TGF-beta1 protein secretion, MMP-2 protein activity and (b). the effect of exogenous TGF-beta1 on MMP-2 protein activity in human lamina cribrosa cells in vitro. METHODS: Primary human lamina cribrosa cells grown on flexible and rigid plates were exposed to cyclical stretch (1Hz, 15%) or static conditions for 12 and 24 hours. Cells grown on 100-mm plates were exposed to human TGF-beta1 (10 ng/ml) or vehicle (4 mM HCl/1% BSA) for 24 hours. TGF-beta1 mRNA synthesis in stretched and static cells was measured using real-time polymerase chain reaction. TGF-beta1 protein secretion in stretched and static cell media was measured using enzyme linked immunosorbent assay. Gelatin zymography measured MMP-2 activity in stretched, static, TGF-beta1- treated and vehicle-treated cell media. RESULTS: Cyclical stretch induced significant increases in TGF-beta1 mRNA synthesis after 12 hours (**P < 0.01) and TGF-beta1 protein secretion after 24 hours (*P < 0.05). Cyclical stretch significantly (*P < 0.05) increased MMP-2 activity in cell media after 24 hours. Exogenous TGF-beta 1 induced a significant (**P < 0.01) increase in cell media MMP-2 activity after 24 hours. CONCLUSIONS: These results suggest that cyclical stretch and TGF-beta1 modulate MMP-2 activity in human lamina cribrosa cells. TGF-beta 1 and MMP-2 release from lamina cribrosa cells may facilitate matrix remodeling of the optic nerve head in primary open angle glaucoma.     

TGF-beta2-induced cell surface tissue transglutaminase increases adhesion and migration of RPE cells on fibronectin through the gelatin-binding domain

PURPOSE: Migration and adhesion of dislocated retinal pigment epithelial (RPE) cells to a fibronectin-rich extracellular matrix is an initial step in proliferative vitreoretinopathy (PVR). In the present study, the functional role of cell surface tissue transglutaminase (tTG) in adhesion and migration of RPE cells on fibronectin (Fn) and collagen type I (Col I) after stimulation with TGF-beta2 was investigated. METHODS: Cultured human RPE cells were treated with 1.0 ng/mL TGF-beta2 for 24 hours. Cell surface tTG expression was determined by cell fraction analysis. Attachment on Col I, full-length Fn, and its 45-kDa gelatin-binding and 110-kDa cell-binding fragment was measured with an MTT assay. Migration of RPE cells was measured by a Boyden chamber assay, and cell spreading was determined. Experiments were performed in the presence or absence of anti-tTG antibodies and anti-integrin alpha5 and beta1 antibodies. RESULTS: TGF-beta2 markedly induced expression of cell-surface tTG on RPE cells and increased attachment and migration on Fn and Col I. Blocking cell surface tTG inhibited attachment, migration, and spreading on Fn and its 45-kDa gelatin-binding fragment, whereas no effect was seen on Col I and the 110-kDa cell-binding Fn fragment. In contrast, blocking of integrin alpha5 and beta1 suppressed adhesion and migration on full-length Fn and the 110-kDa Fn fragment. CONCLUSIONS: These data demonstrate that TGF-beta2 increases expression of cell surface tTG, which in turn strengthens adhesion, migration, and spreading of RPE cells on Fn through the 45-kDa gelatin-binding Fn fragment. At the onset of PVR, this mechanism may help RPE cells to attach and migrate on Fn-containing matrices.     

The Immune Privileged Retina Mediates an Alternative Activation of J774A.1 Cells

Purpose: We have previously found that retinal pigment epithelial (RPE) cells suppressed endotoxin-stimulated macrophages; moreover, it induced expression of anti-inflammatory cytokines. We further assessed the possibility that the RPE is alternatively activating macrophages.

Methods: J774A.1 cells were stimulated with endotoxin and treated with the conditioned media (CM) of RPE, or neuroretinal eyecups from healthy mouse eyes. The supernatant was assayed for IL-1β, TNF-α, IL-6, IL-12(p70), and IL-10, and for nitric-oxide generation. The RPE conditioned media (RPE CM) was absorbed of known soluble factors to identify the factor that augments nitric-oxide generation.

Results: We found the RPE CM suppressed all cytokine production except IL-10, and augmented nitric-oxide generation. The augmented nitric-oxide levels were mediated by RPE derived alpha-melanocyte stimulating hormone (α-MSH).

Conclusions: Healthy RPE not only suppresses inflammatory activity, it promotes an alternative activation of macrophages that can further promote immune privilege.