短发夹RNA对人视网膜色素上皮细胞血管内皮生长因子表达的抑制

目的探讨针对人血管内皮生长因子（VEGF）的短发夹RNA（shRNA）在体外对视网膜色素上皮（RUE）细胞VEGF表达的影响。方法用酶辅助显微分离法分离人RPE细胞,并用细胞角蛋白和S-100进行免疫组化鉴定。设计针对人VEGF的短发夹RNA（shRNAs,P1,P2）,P3为阴性对照即不含特异性shRNA,P1、P2退火后双链DNA连接到质粒pSilencer 4．1-CMV的BamHⅠ和Hjnd Ⅲ双酶切位点。转化细菌后,提取的质粒用EcoRⅠ和SamⅠ进行酶切鉴定。实验分5组,第1组：RPE细胞中在含有100μmol／LCoCl2,10％胎牛血清（FBS）的DMEM培养基中培养30h;第2组：在常规含有10％FBS的DMEM培养基中培养30h;第3、4、5组：分别P1、P2、P3转染细胞24h后在含有100μmoL／LCoCl2的10％FBS的DMEM培养基中培养30h;用免疫印迹法检测各组细胞VEGF表达水平。结果培养的细胞用细胞角蛋白和S-100免疫组化染色阳性。提取的质粒经酶切后片断相对分子质量分别为3300和1600,说明质粒成功地提取和纯化。VEGF表达水平1组明显高于2、3、4组（均P〈0．001）。乏氧（2组与1组比）可以明显增加RPE细胞中VEGF的表达。3、4组（P1和P2）对VEGF表达抑制分别为65．9％和52．4％。5组与1组比差异无统计学意义（P=0．147）。各组间β肌动蛋白表达量差异无统计学意义。结论针对人VEGF的特异性shRNA可以明显降低人RPE细胞VEGF的表达,为RNA干扰治疗新生血管性眼病,尤其是脉络膜新生血管奠定了基础。     

抗血管生成作用的VEGFxxxb在缺氧视网膜色素上皮细胞中的表达

背景 缺氧可诱导视网膜色素上皮（RPE）细胞分泌血管内皮生长因子A（VEGF-A）,从而促进脉络膜新生血管（CNV）形成.VEGF-A依选择性剪接方式的不同,可形成具有促血管生成作用的VEGFxxx和具有抗血管生成作用的VEGFxxxb,但后者在常氧和缺氧状态下在人RPE细胞中的表达变化及其作用尚不清楚.目的 从mRNA水平和蛋白水平检测模拟缺氧状态培养的RPE细胞中VEGFmb的表达,研究VEGFxxxb在常氧和缺氧培养的RPE细胞中的表达变化.方法 以人RPE细胞系ARPE-19为研究对象,将150 μmol/L化学诱导剂CoC12加入细胞培养液制备缺氧细胞模型,分别于0h（常氧培养）及缺氧培养3、6、12和24 h收获细胞或细胞上清液,采用逆转录PCR（RT-PCR）法检测细胞内VEGFxxxmRNA和VEGFxxxb mRNA的表达;采用Western blot法检测细胞内总VEGF-A蛋白和VEGFxxxb蛋白的表达;采用ELISA法检测细胞上清中总VEGF-A蛋白和VEGFxxxb蛋白的含量.结果 在常氧及诱导缺氧后,ARPE-19细胞中均可检测到VEGFxxxmRNA和VEGFxxxb mRNA的表达,检测出的VEGFxxxmRNA中同时出现VEGF121、VEGF165和极微弱的VEGF189条带;检出的VEGFxxxb mRNA中可见VEGF165b条带.缺氧培养的细胞中随着缺氧时间的延长,VEGFxxxmRNA显示出表达逐渐增加的趋势,而VEGFxxxb mRNA则显示出表达逐渐减少的趋势.Western blot法可检测到细胞中VEGFxxxb蛋白的表达,尤以VEGF165b表达最强,随缺氧培养时间的延长,VEGF165b蛋白表达逐渐减少.培养的细胞上清中VEGFxxxb蛋白质量浓度由常氧培养时的（166.82±2.55） pg/ml下降至缺氧培养24 h的（125.35±2.10）pg/ml,而总VEGF-A蛋白则由（294.27±11.97） pg/ml上升至（582.26±12.98） pg/ml.细胞中VEGFxxxbb蛋白占总VEGF-A蛋白的比例随缺氧培养时间的延长,由常氧培养条件下的（56.71±1.02）％逐渐下降至缺氧培养24 h的（21.53±0.08）％.结论 ARPE-19细胞在常氧和缺氧培养条件下均可检测到VEGFxxxbb mRNA和蛋白的?     

视网膜酸化诱导VEGF与PEDF表达及其与氧化应激的关系

背景 缺氧与高糖是引起视网膜新生血管生长的主要原因,可引起视网膜糖酵解作用增强,导致组织酸中毒. 目的 探讨视网膜酸中毒对视网膜血管内皮生长因子(VEGF)与色素上皮衍生因子(PEDF)表达的影响及氧化应激的作用.方法 从2周龄雄性SD大鼠中分离出视网膜.分别在NaHCO3调制的pH7.2、6.8、6.5酸性环境的DMEM培养液中培养24 h;另外,在上述酸性培养液中培养后用PBS洗涤视网膜2遍后置于pH值为7.2的新鲜培养液中继续培养24 h;同时,在上述酸性培养液中加入抗氧化剂对视网膜进行培养.制作视网膜标本,然后行苏木精-伊红染色,采用荧光定量聚合酶链反应( PCR)及免疫蛋白印迹技术检测各组大鼠视网膜中VEGF和PEDF蛋白及其mRNA的表达,以pH7.2作为对照. 结果 视网膜培养24 h后,pH7.2组、pH6.8组视网膜层次清晰,但pH6.5组视网膜出现空泡.正常视网膜VEGF mRNA的表达为(112±11)％,pH7.2组为(100±7)％,差异无统计学意义(P=0.55);pH 6.8组、pH 6.5组中的视网膜VEGF mRNA分别为(196±43)％、(251±29)％,均较pH7.2组明显升高,差异均有统计学意义(P＜0.05).正常视网膜PEDF mRNA水平为(86±19)％,pH7.2组为(100±.33)％,差异无统计学意义(P=0.64);pH 6.5组视网膜PEDF mRNA水平为(230±66)％,较pH7.2组明显升高(P＜0.05).VEGF与PEDF蛋白与其mRNA表达趋势一致.视网膜酸化纠正后,pH 7.2、6.8、6.5组视网膜VEGF mRNA分别为(100±13)％、(111±9)％、(113±9)％,3个组之间比较差异无统计学意义(F=2.51,P=0.16).PEDF mRNA的表达分别为(100±13)％、(110±9)％、(108±11)％,3个组之间比较差异无统计学意义(F=0.98,P=0.43).加入抗氧化剂后,pH7.2组视网膜VEGF mRNA水平为(100±9)％,pH6.8组为(106±7)％,pH 6.5组为(148±22)％,pH6.5组VEGF mRNA表达水平均明显高于pH 7.2组和pH 6.8组(P＜0.05).pH 7.2、6.8、6.5组大鼠视网膜PEDF mRNA表达分别为(100±31)％、(282±45)％、(480±117)％,差异有统计学意义(F=20.73,P=0.00).结论 视网膜酸化诱导VEGF的表达受到氧化应激的调节,抗氧化剂可促进酸化视网膜PEDF的表达增加,表明氧化应激可抑制PEDF表达.     

Choroidal endothelial cells transmigrate across the retinal pigment epithelium but do not proliferate in response to soluble vascular endothelial growth factor

The purpose of this study was to investigate the effects of soluble VEGF on human choroidal endothelial cell (CEC) transmigration across an RPE monolayer as it relates to choroidal neovascularization in AMD. In coculture assays, ARPE-19 (ARPE) was plated on the undersides of Transwell inserts having 0.4 microm pores. Primary human CECs were then plated into the insert. CECs in the Transwell inserts were counted after 72 hr of growth. CEC proliferation was also measured after culturing CECs in ARPE-CEC coculture-conditioned media or in media with exogenous VEGF121 and/or VEGF165 added. Transmigration assays were performed on Transwells with 8.0 microm pores: green-labelled CECs were plated in Transwell inserts with or without red-labelled ARPE plated on the undersides of the insert. In some transmigration assays, ARPE was plated into the wells to provide a chemotactic gradient for CEC transmigration. After 72 hr CECs were plated, green cells were counted either within the well media as CECs that transmigrated the epithelial monolayer, or on the underside of the insert as CECs that transmigrated the Transwell insert to but not beyond the ARPE monolayer. A neutralizing antibody to VEGF was added to the wells of Transwells at the time the CECs were plated in the insert and transmigrated CECs were counted. VEGF protein was measured in the conditioned media of ARPE and CEC coculture and in transmigration assays. Compared to control, CEC proliferation significantly increased when CECs were cultured in coculture conditioned media (p=0.001) or in coculture assays (p<0.001). However, there was no effect on CEC proliferation when VEGF121, VEGF165, or both were added to solo CECs. Antibody to VEGF did not reduce the proliferative effects of coculture conditioned media on CEC. ARPE plated in the well significantly increased CEC transmigration (p<0.001) compared to transmigration assays without ARPE in the well. VEGF protein measured in the well media of transmigration assays having ARPE within the well was significantly greater than in the assays without ARPE within the well (p<0.004). Exogenous neutralizing antibody to VEGF significantly reduced transmigration, and this effect was dose-dependent. VEGF provides a chemotactic gradient for human CECs to transmigrate across a monolayer of ARPE. Neutralization of VEGF in the media partially reduces transmigration. Whereas soluble VEGF does not increase proliferation of solo CECs, coculture conditioned media enhances proliferation, suggesting that growth factors other than VEGF cause CEC proliferation. These findings may have relevance to the transformation of occult CNV into CNV within the neurosensory retina in AMD.     

Geldanamycin, a HSP90 inhibitor, attenuates the hypoxia-induced vascular endothelial growth factor expression in retinal pigment epithelium cells in vitro

Hypoxia is the most common factor contributing to the pathogenesis of choroidal neovascularization, which is the major cause for blindness and occurs in proliferative diabetic retinopathy and age-related macular degeneration. The purpose of this study is to investigate the role of retinal pigment epithelial (RPE) cells in the regulation of subretinal neovascularization under hypoxia and the possible function of a heat shock protein 90 (HSP90) inhibitor, geldanamycin (GA), in the regulation of VEGF expression. An in vitro hypoxic experimental model was used to mimic the ischemic microenvironment of RPE cells. The cell growth was measured by proliferation assay and the morphological observation was documented by microscope. The gene expression of VEGF, hsp70, hsp90alpha and hsp90beta were measured using semi-quantitative RT-PCR. The VEGF release from RPE cells were detected by ELISA. No alteration in growth rate and cell morphology under 1% O(2) condition for 24h was noticed. The proangiogenic growth factor VEGF, but not bFGF, released from hypoxia-treated cells were significantly higher than those of normoxic controls. A similar tendency of VEGF(165) isoform gene expression, detected by RT-PCR, was noticed in hypoxia-treated cells. Heat shock pretreatment elevated hsp70 and VEGF(165) gene expression and augmented the hypoxia-induced VEGF gene expression and protein release. Pretreatment with GA can significantly suppress the hypoxia-induced VEGF gene expression in and peptide release from RPE cells. These in vitro findings suggest that HSP90 inhibitors could be considered as novel anti-angiogenesis agents for diseases with intraocular neovascularization.     

缺氧及血管内皮生长因子对视网膜色素上皮细胞Opticin蛋白表达的影响

目的 探讨缺氧及血管内皮生长因子(VEGF)对视网膜色素上皮(RPE)细胞Opticin 蛋白表达的影响及其内在机制.方法 实验研究.原代培养人眼RPE细胞,取生长良好的第3～6代细胞,接种于含DMEM培养液的6孔板中,于缺氧条件下或加入不同浓度(1、10、50及100 μg/L)VEGF进行培养.逆转录聚合酶链反应(PCR)检测缺氧后RPE细胞中VEGF mRNA的表达.蛋白印迹法检测细胞内和细胞培养液中Opticin蛋白含量.明胶酶谱法检测细胞培养液中明胶酶活性.组间检测数据比较采用单因素方差分析.结果 蛋白印迹法检测,显示缺氧组RPE细胞内Opticin蛋白含量无明显变化;而RPE细胞培养液中,缺氧组Opticin蛋白含量明显下降.逆转录PCR检测,缺氧组12、24 h VEGF mRNA表达灰度值分别为0.81 ±0.04、0.67±0.07,均较正常对照组VEGF mRNA表达灰度值(0.21±0.03)明显升高,差异有统计学意义(F =483.60,P＜0.05).添加不同浓度的外源性VEGF后,RPE细胞内Opticin蛋白表达无明显变化;添加1、10、50及100μg/L的外源性VEGF后,RPE细胞培养液中,Opticin蛋白表达灰度值分别为0.65 ±0.02、0.52±0.04、0.23±0.03、0.30±0.03,均较正常对照组Opticin蛋白表达灰度值(0.73 ±0.04)明显下降(F=141.38,P＜0.05).明胶酶谱法分析,显示与基质金属蛋白酶-2相对应的位置出现明显的消化条带,且酶的活性随VEGF浓度的增加而递增.低浓度的乙二胺四乙酸可抑制由VEGF引起的RPE细胞培养液中Opticin蛋白含量减少.结论 缺氧及VEGF作用可影响RPE细胞培养液中Opticin蛋白分泌,可能与增多的基质金属蛋白酶-2对Opticin蛋白的酶解作用有关.     

高糖对人视网膜色素上皮细胞VEGF及PEDF表达的影响

目的 探讨高糖对人视网膜色素上皮(RPE)细胞表达血管内皮生长因子(VEGF)及色素上皮源性生长因子(PEDF)的干预作用.方法 采用HRPE细胞株,将细胞分为正常对照组(NG,5.6 mmol/L葡萄糖)、高糖组(HG,15、20、30 mmol/L葡萄糖)、高渗组(HM,24.4 mmol/L甘露醇+5.6 mmol/L葡萄糖).应用逆转录PCR(RT-PCR)技术检测VEGF及PEDF mRNA的表达.用ELISA技术检测细胞上清液中VEGF蛋白的表达.结果 高糖作用下VEGFmRNA和蛋白表达显著增高,PEDFmRNA的表达受到显著抑制.结论 高糖可从转录水平及蛋白水平诱导HRPE细胞VEGF的表达,并抑制PEDF的表达.     

Genistein suppressed upregulation of vascular endothelial growth factor expression by cobalt chloride and hypoxia in rabbit retinal pigment epithelium cells.

The time course changes of vascular endothelial growth factor (VEGF) protein expression induced by cobalt chloride (CoCl(2)) and hypoxia and the effects of genistein on CoCl(2)- and hypoxia-induced VEGF expression in rabbit retinal pigment epithelium (RPE) cells were studied. Judged by relative fluorescence using a confocal scanning laser microscope coupled to a computer, VEGF protein expression exposed for different periods to CoCl(2) or hypoxia was investigated. CoCl(2) was found to significantly elevate VEGF protein expression. At 4 h after CoCl(2) treatment, the expression of VEGF protein was about three times as much as that at the start of treatment. Genistein (50, 100 and 200 microM) inhibited VEGF protein expression elicited by CoCl(2) in a concentration-dependent manner. Hypoxia (5% CO(2)/95% N(2)) could markedly increase VEGF protein expression. The elevation of VEGF protein expression was gradual and time-dependently. At 6 h, the highest expression of VEGF protein was observed, it was about three times as much as that at the start of treatment. After preincubation with 50, 100, and 200 microM genistein respectively, the hypoxia-evoked VEGF expression was concentration-dependently suppressed. These results indicated that genistein could be an effective agent in the prevention and treatment of intraretinal and subretinal neovascularization.     

大鼠角膜碱烧伤行培养角膜上皮移植后VEGF的表达

目的 研究角膜上皮培养移植后角膜新生血管(CNV)形成和血管内皮生长因子(VEGF)的变化特点.方法 对角膜碱烧伤CNV大鼠行培养角膜上皮移植,观察术后CNV程度,RT-PCR和免疫组织化学法检测术后24、48、72、168 h角膜组织VEGF的表达.结果 实验组角膜上皮移植后14 d CNV接近瞳孔区,之后逐渐减退.移植后24 h角膜VEGF mRNA的相对含量为1 20±0.09;48 h为1 91±0.03;72 h为2.34±0.11;168 h为1.83 ±0.14.角膜上皮和基质均表达VEGF蛋白,以上皮层表达为主,并随时间推移表达量增加.结论 培养角膜上皮移植后24 h角膜可表达VEGF,72 h表达量最高.VEGF表达与培养角膜上皮移植后CNV的发生密切相关.     

Scleral cell growth is influenced by retinal pigment epithelium in vitro

Background: Studies of form-deprivation myopia in chicks have shown that mitotic activity and the amounts of DNA and protein are increased in the sclera of myopic eyes. Signals acting on the sclera have been speculated to have their origin in the retina, especially in the photoreceptors and the retinal pigment epithelium (RPE). Therefore, in this study we investigated the interaction between the RPE cells and the scleral cells in vitro using chick embryo Methods: At first, we established the method for primary culture of scleral chondrocytes and scleral fibroblasts from chick embryos. Then, we co-cultured scleral chondrocytes with RPE cells and also co-cultured scleral fibroblasts with RPE cells. Scleral cells were cultured on a collagen membrane or Transwell-COL, with RPE cells cultured at the bottom of the dishes. We evaluated the effects of cocultured RPE cells on the proliferation of scleral cells by counting the cell number of scleral cells on those membranes Results: With the co-cultured RPE cells, proliferation of the scleral chondrocytes was inhibited on a collagen membrane, but significantly stimulated on a Transwell-COL. Proliferation of the scleral fibroblasts was also promoted on the Transwell-COL with RPE cells Conclusion: RPE cells influenced the proliferation of scleral cells in vitro, which suggests that RPE may, at least in part, act on the sclera in vivo to regulate ocular growth postnatally.This work was supported by Grant-in-Aid for Scientific Research No. 04454438 from the Ministry of Education, Science and Culture of Japan and by a grant for retinochoroidal atrophy research from the Ministry of Health and Welfare of Japan     

乳酸对鼠视网膜血管内皮生长因子表达的影响

目的 观察不同浓度乳酸对培养的大鼠视网膜血管内皮生长因子(VEGF)表达的影响.方法 2周龄Sprague-Dawley大鼠36只,根据培养液中乳酸含量分为10、20、30 mmol/L乳酸组,每组均为12只大鼠.处死大鼠,摘出眼球剥离视网膜,将视网膜置入插入式培养皿中培养24 h.培养皿中培养液分别为含10、20、30 mmol/L乳酸的Dulbecco改良Eagle培养液+2%胎牛血清.光学显微镜观察视网膜结构、实时荧光定量聚合酶链反应(RT-PCR)和蛋白质免疫印迹(Western blot)检测VEGF的表达.结果 光学显微镜组织病理学观察结果显示,视网膜层次清晰,结构完整,未见明显细胞溶解和坏死.RT-PCR检测结果显示,10、20、30 mmol/L乳酸组VEGF mRNA表达量分别为0.74±0.06、0.99±0.12、1.45±0.17;Western blot检测结果显示,10、20 mmol/L乳酸组和30 mmol/L乳酸组视网膜VEGF表达量分别为0.34±0.15、0.54±0.16、0.93±0.23.RT-PCT和Western blot检测结果均显示30 mmol/L乳酸组较10 mmol/L乳酸组VEGF表达明显升高.结论 乳酸诱导视网膜VEGF表达有浓度依赖性.     

Induction of vascular endothelial growth factor receptor expression in human umbilical vein endothelial cells after repeated bevacizumab treatment in vitro

AIM: To investigate the mechanism underlying the loss of responsiveness to anti-vascular endothelial growth factor (VEGF) treatment after repeated injections for choroidal neovascularization, VEGF and VEGF receptor (VEGFR) expressions were evaluated following repeated bevacizumab treatments in hypoxic human umbilical vein endothelial cells (HUVECs) in vitro. METHODS: HUVECs were incubated under hypoxic conditions in two media of different bevacizumab concentrations (1.0 or 2.5 mg/mL) for 17h, and then in a new medium without bevacizumab for 7h. This procedure was repeated twice more. A culture with an identical volume of excipients served as the control. Cytotoxicity and cell proliferation were assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and Ki-67 assays, respectively. Levels of VEGF and VEGFR were assessed using enzyme-linked immunosorbent assay and Western blot respectively. RESULTS: Cytotoxic effects were not reported for either bevacizumab concentration. Cell proliferation was not reduced after anti-VEGF treatments. VEGF level after single treatment was significantly higher than that of the control and after repeated treatments. Phosphorylated VEGFR-2 expression increased significantly after single and repeated bevacizumab treatments compared with the control. The 1.0 mg/mL bevacizumab induced significantly higher expressions of VEGFR-2 than the 2.5 mg/mL in single and repeated treatment groups. CONCLUSION: Bevacizumab treatment of HUVECs elevated VEGFR expression in both single and repeated treatments, indicating a mechanism for the reduced efficacy of anti-VEGF therapy in ocular neovascular disorders.     

高氧诱导小鼠视网膜病变模型中血管内皮生长因子和色素上皮衍生因子的表达及意义

目的 研究高氧诱导小鼠视网膜病变模型中血管内皮生长因子(VEGF)和色素上皮衍生因子(PEDF)的表达和意义,并探讨两者在视网膜新生血管形成过程中的作用.方法 对照实验研究.对新生C57BL-6N系小鼠给予高氧后,置相对低氧环境中饲养,诱导产生视网膜新生血管.在小鼠生后第12、14及17天摘除眼球,通过逆转录聚合酶链反应和免疫印迹法及荧光素灌注造影,分别检测不同时间点全视网膜新生血管组织对VEGF与PEDF的mRNA和蛋白质的表达水平的差异,并观察视网膜新生血管的分布与形态变化,通过血管内皮细胞计数对新生血管进行量化.应用SPSS11.5统计学软件,采用析因设计方差分析,分别比较实验组与对照组mRNA与蛋白质表达水平的差异,以P<0.05作为差异有统计学意义.结果 在高氧环境中(出生第12天小鼠),OIR模型鼠VEGF蛋白质表达A值(0.47±0.12)较正常鼠(1.81±050)下降,PEDF蛋白质表达A值(5.35±0.94)较正常鼠(0.68±0.17)明显升高;而相对低氧环境中(出生第14和17天小鼠),VEGF蛋白质表达A值(2.15±0.46,5.49±0.97)较正常鼠(0.90±0.05,0.88±0.91)明显升高,PEDF蛋白质表达A值(2.07±0.35,1.37±0.48)较正常鼠(2.62±0.68,5.30±0.59)明显下降,尤以第17天下降显著.对不同时间点VEGF和PEDF蛋白质表达水平进行析因方差分析,结果显示各时间点的VEGF蛋白质表达水平差异有统计学意义(F=70.450,P=0.000),各时间点的PEDF蛋白质表达水平差异有统计学意义(F=160.237,P=0.000).出生第12、14及17天小鼠视网膜VEGF蛋白质的表达与正常对照组比较,差异有统计学意义(P=0.009,0.010,0.000),PEDF蛋白质的表达差异也有统计学意义(P=0.002,0.046,0.000);出生第12、14及17天小鼠视网膜VEGF mRNA的表达与正常对照组比较,差异有统计学意义(P=0.001,0.000,0.001),PEDF mRNA的表达差异也有统计学意义(P=0.000,0.001,0.000).伴随着视网膜组织的缺氧,出现了VEGF蛋白质表达水平升高和PEDF蛋白质表达水平降低,导致视网膜组织中VEGF与PEDF蛋白质表达失衡;VEGF与PEDF的mRNA表达亦发生类似变化,且较蛋白质改变提前发生.上述改变均伴随着同期视网膜新生血管的发生、发展及其严重程度而逐渐加重.结论 VEGF和PEDF的mRNA和蛋白质表达失衡可能足造成视网膜新生血管发生的机制之一.(中华眼科杂志,2008,44:734-740)     

Expression of pigment epithelium derived factor and vascular endothelial growth factor in choroidal neovascular membranes and polypoidal choroidal vasculopathy

AIMS: To determine whether pigment epithelium derived factor (PEDF), a protein that inhibits angiogenesis, is expressed in human choroidal neovascular membranes (CNVMs) and in tissues from an eye with polypoidal choroidal vasculopathy (PCV). In addition, to compare the expression of PEDF with that of vascular endothelial growth factor (VEGF), a known stimulator of angiogenesis, in these tissues. METHODS: CNVMs, associated with age related macular degeneration (AMD), angioid streaks, and PCV, were obtained during surgery. The expression of PEDF and VEGF in the excised subretinal fibrovascular membranes was determined by immunohistochemistry. RESULTS: PEDF and VEGF were strongly expressed in the vascular endothelial cells and retinal pigment epithelial (RPE) cells in the CNVMs where numerous new vessels were prominent (clinically active CNVMs). On the other hand, immunoreactivity for PEDF and VEGF was weak in the new vessels where fibrosis was prominent (clinically quiescent CNVMs). However, the RPE cells were still positive for PEDF and VEGF. The specimens from the eye with PCV also showed strong expression of PEDF and VEGF in the vascular endothelial cells and the RPE cells. CONCLUSION: Because PEDF is an inhibitor of ocular angiogenesis and an inhibitor of ocular cell proliferation, our results suggest that PEDF along with VEGF may modulate the formation of subfoveal fibrovascular membranes.     

Autologous transplantation of retinal pigment epithelium after mechanical debridement of Bruch's membrane

PURPOSE: To determine whether transplantation of autologous retinal pigment epithelium (RPE) will prevent atrophy of the choriocapillaris and loss of photoreceptors in an area in which the RPE has been mechanically debrided from Bruch's membrane. METHODS: Abrasive debridement of RPE was performed with a metal cannula after localized retinal bleb detachments in two separate areas of the rabbit retina. The RPE cell suspension aspirated from one of the debridement sites was transplanted to the other. The debridement-only site served as control. The transplant and control sites were evaluated after 30 days by color fundus photography, fluorescein angiography, light microscopy and transmission electron microscopy. RESULTS: Compared with debridement only, debridement plus transplantation of RPE resulted in more complete repopulation of the bare Bruch's membrane surface with relative preservation of choriocapillaris and photoreceptors. CONCLUSION: Autologous transplantation of RPE onto an abrasively debrided Bruch's membrane decreases choriocapillaris atrophy and photoreceptor loss.