抗血管内皮生长因子小干扰RNA抑制大鼠角膜新生血管形成的研究

目的 探讨抗血管内皮生长因子小干扰RNA(VEGF-siRNA)对角膜碱烧伤急性期新生血管(CNV)形成的抑制作用.方法 实验研究.体外化学合成VEGF序列特异性双链小干扰RNA(VEGF-siRNA),脂质体介导转染体外培养的大鼠角膜上皮和基质细胞,实时PCR法检测转染后各时段VEGF mRNA表达水平,酶联免疫吸附实验测定转染后各时段VEGF蛋白表达水平.制作大鼠角膜碱烧伤动物模型,采用VEGF-siRNA转染的角膜上皮移植联合前房注射脂质体包裹的VEGF-siRNA治疗急性期CNV,免疫组化和ELISA分别检测角膜VEGF表达,观察术后CNV形成.细胞实验和动物实验中siRNA处理组和对照组VEGF蛋白含量比较,采用两样本均数比较的t检验.结果 siRNA对角膜上皮细胞VEGFmRNA的抑制效率达60%～84%,角膜基质细胞VEGFmRNA的抑制效率达59%～76%,角膜上皮和基质细胞VEGF蛋白表达也有相应的下降.采用VEGF-siRNA转染的角膜上皮移植联合前房注射VEGF-siRNA治疗,实验组CNV面积显著小于对照组;实验组VEGF表达水平显著低于对照组.结论 VEGF-siRNA能显著抑制大鼠角膜碱烧伤急性期CNV形成.     

Role of vascular endothelial growth factor in ocular angiogenesis

VEGF-A is a critical regulator of ocular angiogenesis and vascular permeability and is involved in the pathogenesis of several ocular diseases involving neovascularization or increased vascular permeability, such as neovascular AMD, diabetic ME, and diabetic retinopathy. Currently available therapies for neovascular AMD, such as laser photocoagulation, PDT with verteporfin, and pegaptanib sodium, slow visual loss but do not improve vision for most patients. In contrast, an emerging anti-VEGF agent, ranibizumab, improved vision in 25% to 34% of treated patients in one clinical trial, rather than slowing visual loss and is the first treatment for neovascular AMD to demonstrate visual improvement in a substantial number of patients. This represents a major advance in the treatment of ocular diseases involving neovascularization or increased vascular permeability and provides hope to patients with these debilitating diseases. Since the submission of this article, ranibizumab was approved by the FDA for the treatment of neovascular AMD.     

Influence of interferons corneal angiogenesis induced by basic fibroblast growth factor and lipopolysaccharide

A controlled and reproducible angiogenic stimulus was induced in the rabbit cornea by Elvax-40 implants sequestering either 500 ng of lipopolysaccharide (LPS) or 500 ng of basic fibroblast growth factor (bFGF). The effect of IFNα and IFN(β) on angiogenesis was studied by inserting implants sequestering 500 ng of these cytokines (approximately 10(4) Units/implant) adjacent to the LPS or bFGF implants. Interferon-α or γ did not inhibit the bFGF-induced angiogenesis, and in most of these experiments an enhancing effect was observed. This enhancement was not statistically significant. The LPS-induced angiogenesis, however, was slightly inhibited in the presence of either interferon-α or γ but these differences were not statistically significant with p=0.1 only. Both cytokines were non angiogenic and there was no detectable difference between them regarding their effect on the angiogenic process induced by either bFGF or LPS.     

Influence of interferons corneal angiogenesis induced by basic fibroblast growth factor and lipopolysaccharide

A controlled and reproducible angiogenic stimulus was induced in the rabbit cornea by Elvax-40 implants sequestering either 500 ng of lipopolysaccharide (LPS) or 500 ng of basic fibroblast growth factor (bFGF).

The effect of IFNα and IFN_β on angiogenesis was studied by inserting implants sequestering 500 ng of these cytokines (approximately 10⁴ Units/implant) adjacent to the LPS or bFGF implants. Interferon-α or γ did not inhibit the bFGF-induced angiogenesis, and in most of these experiments an enhancing effect was observed. This enhancement was not statistically significant. The LPS-induced angiogenesis, however, was slightly inhibited in the presence of either interferon-α or γ but these differences were not statistically significant with p=0.1 only. Both cytokines were non angiogenic and there was no detectable difference between them regarding their effect on the angiogenic process induced by either bFGF or LPS.     

血管内皮生长因子反义核苷酸抑制大鼠角膜新生血管的实验研究

目的探讨重组腺相关病毒介导的血管内皮生长因子反义核苷酸(rAAV aVEGF165)抑制大鼠角膜新生血管(CNV)发生与发展的作用。方法三质粒共转染法构建rAAV aVEGF165和重组腺相关病毒介导的β半乳糖苷酶的结构基因(rAAV Lacz)。48只SD大鼠用抽签法随机分成实验组和对照组,每组24只,碱烧伤方法诱导CNV生成。实验组结膜囊注射rAAV aVEGF165(1010pfu/ml),对照组结膜囊注射rAAV Lacz(1010pfu/ml);5溴4氯3吲哚βD半乳糖苷(X gal)组织化学染色,检测Lacz基因在结膜囊中的表达情况;碱烧伤后1、2、4、7、14及30d,形态学分析评价角膜新生血管的生长情况,免疫组织化学检测角膜组织中VEGF的表达。结果对照组rAAV Lacz结膜囊注射2d后,检测Lacz基因在结膜组织中的表达效率为21.36%±1.07%;30d后Lacz基因在结膜下组织中的表达效率为28.02%±1.16%。碱烧伤后结膜囊注射rAAV aVEGF165,实验组CNV面积明显减少,新生血管密度降低,VEGF在角膜组织中的表达明显降低,差异有统计学意义(F=1639.22,F=2187.16,F=719.17,P<0.01)。结论rAAV aVEGF165能显著抑制碱烧伤后CNV的形成,其机制与降低角膜组织中VEGF的表达有关,重组腺相关病毒是眼反义基因治疗的有效载体。     

恒河猴血管内皮细胞移植替代角膜内皮细胞后房水血管内皮生长因子水平的变化

目的探索恒河猴血管内皮细胞移植替代角膜内皮细胞后房水血管内皮生长因子(vascular endothelial growth factor,VEGF)浓度的变化。方法实验组将体外培养增殖的恒河猴视网膜-脉络膜血管内皮细胞通过离心沉淀法移植到撕除后弹力层的恒河猴角膜内表面;对照组撕除角膜内皮层后,不做任何处理,直接原位缝合角膜植片。分别于术前及术后1周、2周、3周、4周、6周、8周、12周抽取房水,通过ELISA方法检测其中VEGF浓度,并进行统计分析。结果实验组和对照组术后1周时VEGF浓度差异无统计学意义(P>0.05),术后2周、3周、4周时实验组VEGF浓度分别为(374.74±3.30)ng·L-1、(419.06±1.39)ng·L-1、(481.83±3.36)ng·L-1,同时间点对照组分别为(271.11±1.12)ng·L-1、(345.18±3.27)ng·L-1、(380.33±5.03)ng·L-1,差异均具有统计学意义(均为P<0.05),相应时间点实验组显著高于对照组。实验组术后1周、2周、3周、4周VEGF浓度的差异均有统计学意义(均为P<0.05),各时间点与术前(128.75±3.83)ng·L-1差异均有统计学意义(均为P<0.05)。对照组术后各时间点VEGF浓度与术前差异均有统计学意义(均为P<0.05)。结论血管内皮细胞移植到角膜内表面后,房水VEGF浓度在术后4周内持续升高,之后稳定在一较高水平。     

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。     

瘦素和血管内皮生长因子在碱烧伤诱导的大鼠角膜新生血管模型中的表达

目的 观察碱烧伤后不同时期角膜的组织病理学变化,以及瘦素和血管内皮生长因子(vascular endothelial growth factor,VFGF)的表达,探讨它们与角膜新生血管(corneal neovascularization,CNV)的关系.方法 使用碱烧伤诱导大鼠角膜新生血管模型,采用浸润1 mol·L-1 NaOH溶液、直径为3 mm的单片滤纸,准确置于大鼠右眼角膜中央30 s,制作CNV模型.25只SD大鼠右眼为实验眼,随机分成5组,每组5眼,左眼为正常对照眼.每天用裂隙灯显微镜观察角膜新生血管,分别计算新生血管长度和面积.并行HE染色和免疫组织化学检测.结果 碱烧伤后4 d、7 d、14 d、21 d,实验组CNV面积分别为:(2.55±0.15)m2、(4.15±0.36)mm2、(10.21±0.45)mm2、(8.56±0.06)mm2.免疫组织化学检测显示角膜碱烧伤后1 d,实验组瘦素、VEGF的表达开始增高,碱烧伤后14 d达高峰,14 d后瘦素、VEGF的表达下降,21 d后显著下降;其表达部位主要分布在形成的新生血管区域和上皮层;正常对照组瘦素可微弱表达于角膜上皮层和基质层,VEGF没有表达或仅在上皮基底膜有微弱表达.实验组角膜瘦素、VEGF阳性表达率较对照组明显增加(χ2=29.07,P=0.001;χ2=35.51,P=0.001),瘦素与VEGF的表达具有正相关性(r=0.95,P=0.001).结论 瘦素和VEGF表达水平与角膜新生血管的生成具有相关性,瘦素可能成为治疗CNV的靶点.     

二十碳五烯酸抑制血管内皮生长因子诱导人血管内皮细胞增生作用的研究

背景研究表明二十碳五烯酸（EPA）作为主要的脂质参与人体的生物代谢活动,抑制血管内皮生长因子（VEGF）的产生、阻滞血管平滑肌细胞的内移和增生。而眼部多种疾病与新生血管的形成有关。目的探讨EPA对VEGF诱导的人脐静脉内皮细胞（UVEC）增生的抑制作用。方法对人UVEC株培养和传代,并用Ⅷ因子相关抗原多克降抗体进行免疫细胞化学鉴定。应用免疫组织化学法检测EPA对VEGF受体2（Flk-1）在人UVEC中表达的影响;用MTT比色法检测不同质量浓度EPA作用不同时间后对非VEGF诱导及VEGF诱导的人UVEC增生的抑制率;用流式细胞术检测EPA对VEGF诱导的人UVEC细胞周期的影响。结果培养和传代的人UVEC呈纺锤形及鹅卵石样排列,Ⅷ因子相关抗原鉴定呈阳性反应。EPA作用后,Flk-1在人UVEC中的阳性染色强度明显弱于对照组,Flk-1阳性细胞数明显减少。6个质量浓度组的EPA对VEGF诱导及非VEGF诱导的人UVEC的增生均有明显的抑制作用,其作用呈质量浓度依赖性（F：23．072,P=0．000）;各质量浓度组的EPA对VECF诱导的人UVEC的抑制作用强于非VEGF诱导的人UVEC,差异有统计学意义（F=41．417,P=0．000）。各质量浓度组EPA对VEGF诱导的人UVEC作用24、48、72h后,其细胞数逐渐降低（F=87．823,P=0．000）,但作用时间对其抑制作用无明显影响（F=1．495,P=0．236）。EPA使VEGF诱导的人UVEC细胞阻滞在G0／G1期,EPA组G0／G1期细胞数比例为（75．83±1．56）％,对照组为（68．62±1．44）％,差异有统计学意义（t=-5．88,P=0．00）;EPA组与对照组均未发现凋亡细胞。结论EPA能够通过阻止人血管内皮细胞的DNA合成而明显抑制VEGF诱导的人UVEC的增生,其抑制作用呈剂量依赖性;EPA作用后人UVEC中Flk-1的表达下调,提示EPA可能有抗血管生成的作用。     

Intravitreal vascular endothelial growth factor

Purpose

To evaluate whether a specific pre-analytical stabilization regimen is needed for naïve vitreous taps to detect true values of intrinsic VEGF levels.

Methods

Fourteen consecutive patients with different vitreomacular pathologies without blood–retina-barrier breakdown were scheduled for standard 23-gauge three-port pars plana vitrectomy, and naïve vitreous taps were sampled at the beginning of each procedure. The extracted vitreous specimen was split; one half was immediately stored in a ?20 °C freezer (unstabilized samples) and the other half was instantly stabilized with albumin (2.5 % final conc.), followed by arginine stabilization (1.25 M final conc.) and consecutively stored in a ?20 °C freezer (stabilized samples).

Results

Intravitreal VEGF was detected in all 14 analyzed samples (100 %). VEGF levels were shown to be 46.5 pg/ml?±?62.3 pg/ml (MV ± SD; range: 5.99–232.3 pg/ml) in unstabilized, and 120.4 pg/ml?±?94.4 pg/ml (range: 42.9 pg/ml–289.6 pg/ml) in stabilized vitreous samples. Intravitreal VEGF levels in stabilized vitreous samples were on average 2.6-fold, and thus significantly higher than in unstabilized taps of same eyes (p?=?0.001, Wilcoxon test). VEGF levels in stabilized vitreous samples can be up to 8.5 times higher than in corresponding unstabilized vitreous taps of same eyes (bootstrap analysis). Intravitreal VEGF levels in unstabilized samples correlate with those in stabilized vitreous taps (r?=?0.594; p?=?0.025; Pearson).

Conclusions

An adequate pre-analytic stabilization regimen is needed to evaluate the most accurate intravitreal VEGF levels. This in turn will result in a better understanding of the physiological as well as pathological role of VEGF within the eye. Furthermore, knowing the true value of intravitreal VEGF levels will help to calculate the dosage of intravitrealy applied anti-VEGF drugs.     

角膜碱烧伤后VEGF的表达与新生血管的关系

目的研究碱烧伤后角膜血管内皮生长因子（vascular endothelial growthfactor，VEGF）的表达与角膜新生血管化的关系。方法30只Sprague-Dawleg（SD）大鼠碱烧伤，诱导角膜新生血管模型。碱烧伤后不同时间形态学分析来评价角膜新生血管的情况，免疫组化及Westem blot评价角膜VEGF的表达。结果角膜碱烧伤后24hVEGF的表达开始增高，伤后4d达高峰，伤后7d VEGF的表达下降，14d后显著下降。碱烧伤后，角膜新生血管与VEGF的表达呈明显的平行关系。结论碱烧伤后大鼠角膜VEGF的表达水平与新生血管的形成有相关性，并在其中发挥重要作用。     

Bevacizumab inhibits corneal neovascularization in an alkali burn induced model of corneal angiogenesis

BACKGROUND: New and uncontrolled blood vessel development in the cornea is a pivotal process in the pathogenesis of several corneal diseases. These corneal diseases may finally cause blindness and managing them therapeutically is problematic. The data supporting a causal role for vascular endothelial growth factor in corneal neovascularization are extensive. This study aimed to evaluate the effect of subconjunctival bevacizumab (Avastin) on experimental corneal neovascularization in rabbits. METHODS: Chemical cauterization of the cornea was performed by touching central cornea with a 5-mm-diameter NaOH-soaked cotton applicator for 10 s in 20 eyes of 20 White New Zealand rabbits. The rabbits were then divided randomly into two equal groups. Bevacizumab (2.5 mg) was administered to 10 eyes (group 1) by a subconjunctival injection immediately after chemical cauterization of corneal surface. As a control, 10 eyes (group 2) received an injection of distilled water. Rabbits were examined daily for detection of the first signs of neovascularization. Three weeks later, the extent of corneal neovascularization was evaluated by direct examination and photograph analyses. Total corneal neovascularization area, degree of circumference involved and longest neovascular pedicle length were assessed. RESULTS: Bevacizumab significantly decreased the total neovascularization area (P < 0.009), the circumference involved (P < 0.011) and the longest neovascular pedicle length (P < 0.023). CONCLUSION: Local injection of bevacizumab has a significant effect on inhibition of alkali burn-induced corneal neovascularization. This shows the potential value of bevacizumab in the treatment of corneal neovascularization.     

Soluble vascular endothelial growth factor receptor-1 contributes to the corneal antiangiogenic barrier

PURPOSE: Pathological neovascularisation within the normally avascular cornea is a serious event that can interfere with normal vision. Upregulation of vascular endothelial growth factor (VEGF) has been associated with neovascularisation in the eye, suggesting that maintaining low levels of VEGF is important for corneal avascularity and intact vision. This study aims to determine the expression profile and possible contribution of sVEGFR-1 to the corneal avascular barrier. DESIGN: Experimental case series investigating VEGF and soluble fms-like tyrosine kinase (sFlt) levels in normal and neovascularised human corneas. PARTICIPANTS: Four normal human corneas, five human corneas with alkali burns, three human corneas with aniridia, one with ocular cicatricial pemphigoid and two with interstitial keratitis were examined. METHODS: Western blot and immunohistochemical analyses were performed to determine sFlt and VEGF levels in normal and neovascularised human corneas. Immunoprecipitation was utilised to demonstrate sFlt-VEGF binding. RESULTS: Normal human corneas strongly express sFlt in the corneal epithelium and weakly in the corneal stroma close to the limbus. VEGF is bound by sFlt in the normal human cornea. Neovascularised human corneas have greatly reduced expression of sFlt and significantly less VEGF bound by sFlt. CONCLUSIONS: sFlt is highly expressed in the human cornea and normally sequesters VEGF.     

miR-191抑制视网膜血管内皮细胞增生和血管形成

目的:观察慢病毒（LV）介导miR-191对体外培养的人视网膜血管内皮细胞（hREC）增生和血管生成的抑制作用。方法:体外培养hREC细胞系,分为对照组、缺氧组、LV-空载体（LV-vector）组、LV-miR-191 （LV-191）组。LV-vector组、LV-191组分别转入相应的慢病毒载体。采用流式细胞仪检...     

Natural history of choroidal neovascularization induced by vascular endothelial growth factor in the primate

Background: A new model of choroidal neovascularization (CNV) has been developed in the primate by implanting vascular endothelial growth factor (VEGF)-impregnated microspheres in the subretinal space. Methods: CNV was induced in Macaca mulatta monkeys by implanting VEGF-impregnated gelatin microspheres in the subretinal space. Progression of CNV was followed for 24 weeks after surgery using fluorescein angiography. Eyes were enucleated at various time points, and lesions were evaluated for evidence of CNV by light microscopy and by immunohistochemical staining. Results: CNV developed in 12 (92%) of 13 eyes. Fluorescein leakage was first observed in the 2nd postoperative week and was apparent for the following 12 weeks. CD31 staining for endothelial cells was first observed at day 7 and was evident for the following 8 weeks. Glial fibrillary acidic protein staining revealed a glial adhesion between the proliferative membrane and the retina at 6 weeks after implantation. Smooth muscle actin-positive cells were found a + 2 weeks and remained prominent for at least the next 6 weeks. Cytokeratin-positive retinal pigment epithelial (RPE) cells, first identified in the proliferative membrane at day 3, predominated throughout the growth of the membrane. Macrophages (RAM-II positive) were present at day 3 but were no longer observed after day 7. Conclusion: In monkeys, subretinal implantation of VEGF-impregnated gelatin microspheres leads to the development of CNV. Early, disciform and reparative stages of CNV were observed, similar to those seen in humans. This model will be useful for studying the pathogenesis of CNV and for evaluating potential treatment strategies. Received: 7 June 1999 Accepted: 9 September 1999     

Vitreous levels of pigment epithelium-derived factor and vascular endothelial growth factor: implications for ocular angiogenesis

PURPOSE: Pigment epithelium-derived factor (PEDF) has been demonstrated to suppress ocular angiogenesis in several animal models. In this study, we sought to measure the levels of PEDF and vascular endothelial growth factor (VEGF) in the vitreous of patients with and without ocular neovascular disorders. DESIGN: Case-control study of patients undergoing intraocular surgery for a variety of neovascular and nonneovascular conditions. METHODS: Vitreous samples were collected from 65 eyes of 65 patients with no neovascular disorder (n = 24), choroidal neovascularization (n = 9), active proliferative diabetic retinopathy (n = 16), and inactive proliferative diabetic retinopathy (n = 16). The levels of VEGF and PEDF in these vitreous samples were determined by enzyme-linked immunosorbent assay. RESULTS: The VEGF levels were at or below the level of detectability in the reference and choroidal neovascularization groups. The VEGF levels were significantly elevated in both the active and inactive PDR groups, and significantly higher in the active PDR group as compared with the inactive PDR group. The PEDF levels, which were present at relatively high concentrations in all groups, were higher in patients with active PDR compared with the control and choroidal neovascularization groups. CONCLUSIONS: High levels of immunoreactive PEDF are present in the vitreous of individuals with or without ocular neovascularization, but PEDF levels are significantly higher in patients with active PDR compared with patients with choroidal neovascularization or nonneovascular retinal diseases. Although these results do not preclude the possibility that endogenous PEDF helps to modulate ocular neovascularization, they do not support ischemia-induced downregulation of PEDF as a mechanism for such modulation.