血管内皮生长因子及其受体在实验性脉络膜新生血管中的表达

目的 探讨血管内皮生长因子(VEGF)及其FLK1受体在氪激光诱导的棕色挪威(BN)大鼠脉络膜新生血管(CNV)中的表达。方法 应用氪激光对30只雄性BN大鼠实验眼进行视网膜光凝,创建CNV模型,分别于光凝后3、7、14、21、28及56 d行荧光素眼底血管造影(FFA),处死大鼠后摘除眼球,制作组织病理学标本观察,原位杂交检测VEGFmRNA,免疫组化检测VEGF和FLK1受体。结果 正常BN大鼠视网膜神经节细胞层、内核层、色素上皮层、视网膜及脉络膜血管内皮细胞中均表达VEGFmRNA。实验眼光凝后3 d,光凝区视网膜神经节细胞层、内核层、外核层缺损区、视网膜及脉络膜血管内皮细胞均表达VEGFmRNA,此时视网膜下未形成CNV;3-21 d视网膜内VEGFmRNA表达水平逐渐下降(P<0.01);21 d与28 d和56 d比较,VEGFmRNA表达水平差异无显著意义(P>0.05)。光凝后7 d,FFA检查可见光凝区有圆盘状荧光素渗漏。病理切片可见视网膜下形成CNV,CNV中VEGFmRNA表达阳性;7～21 d,CNV中VEGFmRNA阳性染色面积及吸光度(A)值逐渐增加(P<0.01);21 d后VEGFmRNA表达水平差异无显著意义(P>0.05)。正常视网膜和脉络膜血管内皮细胞及视网膜神经节细胞层FLK1受体表达阳性;光凝后7 d,CNV中FLK1受体表达阳性;随CNV的增生,FLK1受体阳性染色面积及A值逐渐增加(P<0.01);21 d与28 d和5     

Nicotine promotes contribution of bone marrow-derived cells to experimental choroidal neovascularization in mice

Nicotine can increase size and severity of experimental choroidal neovascularization (CNV); however, the mechanism is uncertain. Recent studies demonstrated that the development of CNV involves the contribution of bone marrow-derived cells (BMCs). This study aims to investigate the effects and the potential mechanism of nicotine on BMCs' contribution to CNV. Green fluorescent protein (GFP) chimeric mice were developed by transplanting bone marrow cells from GFP transgenic mice to C57BL/6J mice. CNV was induced by lasering. Nicotine was administered orally in drinking water. Histopathologic study and choroidal flatmount were performed to measure the CNV severity and BMCs recruitment. BMCs expressing different cell markers in CNV and local expressions of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and vascular cell adhesion molecule-1 (VCAM-1) were detected by immunofluorescence. Nicotine administration resulted in larger diameter and surface area of CNV (P<0.05). Nicotine-exposed mice demonstrated increased area and density of GFP+ cells, increased GFP+ vascular cells area, and decreased ratio of BMCs expressing F4/80 in CNV (P<0.05). Furthermore, the expression of VEGF and bFGF within CNV and VCAM-1 in choroid beneath CNV was up-regulated in nicotine-exposed mice. Our results suggest that nicotine promotes recruitment and incorporation of BMCs into CNV and affects differentiation of BMCs in CNV. These effects may be partly due to indirect actions of nicotine on BMCs via other factors (e.g. VEGF or VCAM-1). It is helpful to understand the mechanism of the effect of nicotine in CNV development.     

康柏西普对实验性脉络膜新生血管的作用机制研究

目的　探究康柏西普对实验性脉络膜新生血管（choroidal neovascularization，CNV）的作用机制。方法　选取40只7周龄的雄性BN大鼠，采用多波长氪激光对豚鼠左眼行视网膜光凝，制作CNV大鼠模型，并随机分为4组，空白对照组、模型组、低剂量康柏西普组和高剂量康柏西普组；于造模后7 d、14 d注射康柏西普，并在造模后21 d进行右眼眼底彩照、眼底荧光素血管造影（fundus fluorescein angiography，FFA）检查，检测CNV 发生率及渗漏面积，使用FITC灌注脉络膜铺片测量CNV面积，使用HE染色观察大鼠视网膜结构，使用Western blot检测大鼠视网膜中血管内皮生长因子（vascular endothelial growth factor，VEGF）、缺氧诱导因子-1α（hypoxia-inducible factor-1α，HIF-1α）、丝氨酸/苏氨酸激酶 （serine/threonine kinase，AKT）表达情况。结果　除吲哚菁绿血管造影荧光点数、CNV发生率外，相比空白对照组，模型组大鼠CNV渗透面积，荧光素渗漏面积，脉络膜结构损伤程度，视网膜中 HIF-1α、AKT和VEGF蛋白的表达均显著升高，差异均有统计学意义（均为P<0.05）；相比模型组，康柏西普组大鼠上述各指标显著降低，高剂量康柏西普组上述各指标显著低于低剂量康柏西普组，差异均有统计学意义（均为P<0.05）。结论　康柏西普可以抑制脉络膜视网膜中 HIF-1α、AKT和VEGF蛋白的表达，从而抑制由氪激光光凝眼底而产生的实验性CNV生长。     

实验性脉络膜新生血管中环氧合酶-2、血管内皮生长因子和基质金属蛋白酶-2的表达

目的研究实验性脉络膜新生血管（CNV）大鼠中环氧合酶-2（COX-2）、血管内皮生长因子（VEGF）和基质金属蛋白酶-2（MMP-2）的表达,并进一步探讨其在CNV中的作用机制及相互关系。方法应用氪红激光光凝视网膜的方法诱导制作BN大鼠的CNV模型,分别选取并制作无任何干预的对照组和实验组光凝后第3、7、14、21、30天的＂最大CNV膜＂切片,采用免疫组织化学技术检测COX-2、VEGF和MMP-2在视网膜和CNV膜中的表达。应用HPIAS-1000型高清晰度彩色病理图文分析系统测定COX-2、VEGF和MMP-2阳性染色的平均灰度值。结果 COX-2、VEGF和MMP-2在正常视网膜和脉络膜中呈弱表达,视网膜光凝后,COX-2、VEGF和MMP-2在视网膜和CNV膜中的表达在7～14 d逐渐增强,21 d时三者的表达强度均达到高峰,之后略减弱。视网膜光凝后7、14、21、30 d,实验组COX-2、VEGF和MMP-2的免疫组织化学染色平均灰度值与对照组相比差异均有统计学意义（P〈0.05）。COX-2的表达强度与VEGF和MMP-2的变化均呈正相关（r=0.967,P=0.007;r=0.966,P=0.007）。结论激光诱导的实验性CNV中COX-2、VEGF及MMP-2的表达随着时间的延长呈动态变化,CNV局部炎症诱导的COX-2表达可能是VEGF和MMP-2的上游调节因子。     

Regression of ocular neovascularization in response to increased expression of pigment epithelium-derived factor

PURPOSE: Several pharmacologic treatments have been shown to reduce ocular neovascularization when administered before the onset of angiogenic stimuli, but none have been shown to cause regression of already established ocular neovascularization. In this study, the authors tested the effect of adenoviral vectored pigment epithelium-derived factor (PEDF) gene transfer on established neovascularization in transgenic mice with expression of vascular endothelial growth factor (VEGF) in photoreceptors (rho/VEGF mice) and in a model of choroidal neovascularization. METHODS: Two weeks after the onset of VEGF transgene expression in rho/VEGF mice or 2 weeks after laser-induced rupture of Bruch's membrane in wild-type mice, subgroups of mice were killed, and the baseline amount of neovascularization was measured by image analysis. The remainder of the mice received an intravitreous or subretinal injection of adenoviral vector containing a PEDF expression construct (AdPEDF.11) or control vector (AdNull.11). RESULTS: Seven days after injection in rho/VEGF mice or 10 days after injection in the choroidal neovascularization model, the amount of neovascularization in AdPEDF.11-injected eyes was significantly less than the baseline level, indicating that regression of neovascularization had occurred. There was TUNEL staining within choroidal neovascular lesions in eyes injected with AdPEDF.11. Eyes given a subretinal injection of AdNull.11 had TUNEL-positive cells in the retina, but none within areas of choroidal neovascularization. CONCLUSIONS: These data indicate that increased expression of PEDF causes regression of ocular neovascularization by promoting apoptosis of cells within neovascular lesions and possibly represents a new treatment paradigm for patients with established ocular neovascularization.     

骨髓来源细胞在脉络膜新生血管生成中的作用

目的 探讨骨髓来源细胞(BMCs)在脉络膜新生血管(CNV)生成中的作用。 方法 将绿色荧光蛋白(GFP)转基因小鼠骨髓细胞移植给野生型C57BL/6J小鼠建立嵌合体,应用532 nm倍频激光诱发嵌合体小鼠(处理组)和野生型小鼠(对照组)CNV。利用脉络膜铺片观察由GFP阳性细胞形成的新生血管;免疫荧光染色观察CNV中GFP 阳性细胞是否表达血管内皮生长因子(VEGF)和碱性成纤维细胞生长因子(bFGF)。结果 激光光凝后第29天,脉络膜铺片显示CNV中有大量绿色的骨髓来源细胞,由其组成的血管面积约占CNV表面积的16.22%;免疫荧光染色结果显示,CNV区域的一些BMCs表达VEGF和bFGF。结论 BMCs通过参与新生血管组成及分泌促血管生成因子的方式,可能在CNV发生过程中发挥着重要作用。     

Intravitreal triamcinolone does not alter basal vascular endothelial growth factor mRNA expression in rat retina

Intravitreal triamcinolone inhibits choroidal neovascularization. To investigate if vascular endothelial growth factor (VEGF) is affected, we examined VEGF expression after intravitreal triamcinolone administration in rat retina. Using in situ hybridization, we have found dense clustered VEGF mRNA signals in the retinal pigment epithelium, moderate patchy signals in the inner nuclear layer, and positive labeling in a sub-population of ganglion cells. Densitometry and northern blot analysis revealed no significant alteration of VEGF mRNA expression pattern and level 3-21 days after triamcinolone injection. Our data indicate that intravitreal triamcinolone does not affect basal VEGF mRNA expression in normal adult rat retina.     

Expression of endoglin in choroidal neovascularization

Endoglin (CD105) is a membrane protein involved in the TGF-beta receptor signalling pathway with predominant expression by proliferating endothelial cells. The aim of this study is to analyze the expression of Endoglin in choroidal neovascularization membranes (CNVM) and to compare it to the overall proliferative status of CNVM. Thirty surgically excised CNVM, secondary to age-related macular degeneration, were investigated using light microscopic immunohistochemistry and confocal immunofluorescence microscopy using verified antibodies directed against the endothelial cell markers Endoglin, von Willebrand factor (vWF) and CD34 and the proliferation marker Ki-67. Donor eyes were used as controls. A selective expression of CD34 and vWF as well as Endoglin was found in endothelial cells. Endoglin expression was elevated in vascular endothelial cells contained within CNVM, but a moderate Endoglin expression could also be visualized in quiescent CD34 and vWF positive ocular vasculature. Ki-67 positive cells were detected in CNVM, but these were rarely endothelial cells. Endoglin expression in endothelial cells of CNVM is increased, but rarely associated with a concomitant expression of the proliferation marker Ki-67. The elevated expression of Endoglin in surgically excised CNVM suggests a persisting post-mitotic activation in an advanced stage of this neovascular tissue.     

Expression of pigment epithelium-derived factor in normal adult rat eye and experimental choroidal neovascularization

PURPOSE: Pigment epithelium-derived factor (PEDF) is a protein produced by the retinal pigment epithelial (RPE) cells. Recent studies have implicated PEDF in activities that are inhibitory to angiogenesis. In this study, the expression of PEDF was investigated in normal rat eyes and in eyes with experimentally induced choroidal neovascularization and compared with the expression of vascular endothelial growth factor (VEGF). METHODS: Choroidal neovascularization was induced by laser photocoagulation in rat eyes. At intervals of up to 2 weeks after photocoagulation, the eyes were removed and prepared for in situ hybridization and immunohistochemical study. In situ hybridization was performed with digoxigenin-labeled PEDF riboprobes. Protein expression of PEDF and VEGF was studied immunohistochemically. RESULTS: In normal adult rat eyes, PEDF mRNA was observed mainly in the corneal epithelial and endothelial cells, lens epithelial cells, ciliary epithelial cells, retinal ganglion cells, and the RPE cells. During the development of choroidal neovascularization, PEDF mRNA, PEDF protein, and VEGF protein were strongly detected in many cells within the laser lesions at 3 days after photocoagulation, after which levels gradually declined. However, PEDF was still expressed in the RPE cells that proliferated and covered the neovascular tissues at 2 weeks, whereas VEGF protein was weakly expressed in endothelial cells in choroidal neovascularization. CONCLUSIONS: PEDF is expressed in different cell types of normal rat eyes. The expression of PEDF was detected in the choroidal neovascular tissues induced by photocoagulation, and these findings suggest that PEDF may modulate the process of choroidal neovascularization.     

Expression of connective tissue growth factor and its potential role in choroidal neovascularization

BACKGROUND: To determine the expression of connective tissue growth factor (CTGF) in choroidal neovascularization. METHODS: Surgically excised choroidal neovascular membranes (CNVMs) were obtained at vitrectomy from eight eyes with age-related macular degeneration, five eyes with high myopia, and two eyes with angioid streaks. Light microscopic immunohistochemical analysis was performed to detect CTGF, transforming growth factor beta 1 (TGF-beta1), vascular endothelial growth factor (VEGF), pancytokeratin, and smooth muscle actin (SMA). RESULTS: CNVMs were classified by fibrotic status as cellular CNVM, moderate fibrous CNVM, and extensive fibrous CNVM. CTGF expression was found in vascular cells, stromal cells, and retinal pigment epithelium (RPE) cells. For the stromal cells, fibroblastlike cells were most strongly positive for CTGF. CTGF immunoreactivity in the stroma was stronger in the fibrous CNVMs than in the cellular CNVMs. Immunohistochemical analysis of serial sections revealed colocalization of CTGF with TGF-beta1 and VEGF; colocalization of CTGF with pancytokeratin and SMA was also found. CONCLUSION: Our findings suggest that transdifferentiated RPE cells and vascular cells possess remarkable CTGF expression in CNVMs. This expression of CTGF may stimulate fibroblasts to produce extracellular matrix and may promote angiogenesis in vascular cells. Colocalized TGF-beta1 and VEGF may also contribute the upregulation of CTGF.     

Inhibition of choroidal neovascularization by lentivirus-mediated PEDF gene transfer in rats

AIM: To evaluate the effects of lentivirus-mediated pigment epithelium-derived factor (PEDF) gene transfer performed in treatment of rats with established choroidal neovascularization (CNV), and investigates the mechanism by which PEDF inhibits CNV in rats. METHODS: Brown Norway (BN) rats (n=204) were induced by exposure to a laser, and then randomly assigned to 3 groups: no treatment; treatments with intravitreal injection of lentivirus-PEDF-green fluorescent protein (GFP) or lentivirus-control GFP (free fluorescent protein). Following induction and treatment, the CNV tissue was assessed for form, size and vessel leakage by fluorescein fundus angiography (FFA), optical coherence tomography (OCT), histopathology, and examination of choroidal flat mounts. VEGF, Flk-1, and PEDF expression were evaluated by real-time polymerase chain reaction (PCR) and Western blot. RESULTS: A stable laser-induced rat model of CNV was successfully established, and used to demonstrate lentivirus-mediated PEDG gene transfer by intravitreal injection. Expression of green fluorescence labelled PEDF was observed in the retina up to 28d after injection. An intravitreal injection of lentivirus-PEDF-GFP at 7d led to a significant reduction in the size, thickness and area of CNV showed by FFA, OCT and choroidal flat mounts. PEDF was up-regulated while VEGF and Flk-1 were down-regulated in the lentivirus-PEDF-GFP group. The differences in VEGF and Flk-1 expression in the control and lentivirus-PEDF groups at 7, 14, 21 and 28d after laser induction were all statistically significant. CONCLUSION: Lentivirus-mediated PEDF gene transfer is effective for use in treatment of laser-induced CNV, and PEDF exerts its therapeutic effects by inhibiting expression of VEGF and Flk-1.     

PEDF和VEGF mRNA在实验性脉络膜新生血管组织中的表达

目的 研究血管内皮生长因子(vessel endothelial growth factor，VEGF)和色素上皮衍生因子(pigment epithelium derived factor，PEDF)在实验性小鼠脉络膜新生血管(choroidal neowascularization,CNV)组织中的表达情况。探讨二者在CNV形成过程中所起的作用。方法 用半导体激光诱导小鼠CNV模型。分别于激光后1、3、7d、2和3周时取出眼球，采用原位杂交方法检测CNV组织中VEGF和PEDF mRNA的表达情况。结果 VEGF和PEDF mRNA在激光诱导的小鼠CNV组织形成过程中均有显著表达。激光光凝早期二者的表达均增高，但VEGF mRNA的表达升高更显著。激光照射后3和7d时．VEGF mRNA的表达即达到高峰，阳性率分别为26．05％和27．92％，而PEDF mRNA的阳性表达率分别为21．13％和23．55％．2周时，VEGF mRNA表达开始下降，约为23．95％，而PEDF mRNA的表达则达到高峰,为29．19％,光凝后3周时，二者的表达均下降，但PEDF mRNA的表达仍高于VEGF mRNA的表达，分别为24．87％和21．93％．结论 VEGF和PEDF mRNA明显表达于实验性小鼠CNV组织中。2者表达失衡可能在CNV的形成过程中起到调控作用。     

兔视网膜静脉阻塞眼视网膜新生血管组织中 血管内皮生长因子mRNA的表达

目的探讨血管内皮生长因子(vascular endothelial growth factor,VEGF )在视网膜新生血管组织的发生发展中的参与机制。方法 采用氩激光直接光凝法封闭兔眼视网膜静脉建立视网膜静脉阻塞(retinal vein occlusion,RVO)模型。利用组织原位杂交法观察缺血视网膜组织及新生血管组织中VEGF mRNA的表达。结果缺血视网膜及新生血管组 织中有不同程度的VEGF mRNA表达,表达的程度以视网膜新生血管组织中最强。 VEGFmRNA 的表达部位与视网膜组织缺血的分布具有一定的对应性。结论VEGF在视网膜血管增生性病变的发生中可能具有重要的参与机制。(中华眼底病杂志,2001,17:5-7)     

经瞳孔温热疗法照射对视网膜组织中VEGF和MMP-1 mRNA表达的影响

目的研究经瞳孔温热疗法（transpupillary thermotherapy,TTT）照射对视网膜组织中血管内皮生长因子（vascular endothelial growth factor，VEGF）和基质金属蛋白酶-1（matrix metalloproteinase-1，MMP—1）表达的影响。方法使用唧照射正常灰免视网膜．激光参数为：3min光斑直径，1min曝光时间，450mW功率。每眼照射15点。于照射后1d、3d、7d、2周和3周处死动物并取视网膜组织。抽提RNA后使用RT-PCR方法进行扩增。结果与正常视网膜相比，TTT照射后1d和3d时VEGF mRNA表达降低，7d时恢复正常；MMP-1 mRNA在TTT照射后升高，持续至2周，3周时明显下降。结论TTT照射可能通过降低视网膜组织VEGF的表达来起到抑制新生血管的作用．TTT照射引起MMP-1的表达升高可能通过增加胶原成分的降解来促进新生血管纤维膜的回退。     

Combretastatin A-4 phosphate suppresses development and induces regression of choroidal neovascularization

PURPOSE: Combretastatin A-4 (CA-4) is a naturally occurring agent that binds tubulin and causes necrosis and shrinkage of tumors by damaging their blood vessels. In this study the effect of a CA-4 prodrug, combretastatin A-4-phosphate (CA-4-P), was tested in two models of ocular neovascularization. METHODS: The effect of CA-4-P was quantitatively assessed in transgenic mice with overexpression of vascular endothelial growth factor in the retina (rho/VEGF mice) and mice with choroidal neovascularization (CNV) due to laser-induced rupture of Bruch's membrane. RESULTS: In rho/VEGF mice, daily intraperitoneal injections of 4.0 mg/kg CA-4-P starting at postnatal day (P)7, the time of onset of transgene expression, resulted in a significant reduction in the number of neovascular lesions and total area of neovascularization per retina at P21, compared with vehicle-injected mice. In mice with laser-induced rupture of Bruch's membrane, daily intraperitoneal injections of 75 or 100 mg/kg CA-4-P resulted in a significant reduction in the area of CNV at rupture sites compared with vehicle-injected mice. In mice with established CNV, daily intraperitoneal injections of 100 mg/kg CA-4-P for 1 week resulted in a significant reduction in CNV area at rupture sites compared with the baseline area before treatment or the area of CNV in vehicle-treated mice. CONCLUSIONS: These data indicate that CA-4-P suppresses the development of VEGF-induced neovascularization in the retina and both blocks development and promotes regression of CNV. Therefore, CA-4-P shows potential for both prevention and treatment of ocular neovascularization.     

洛伐他汀对大鼠角膜新生血管VEGF和TGF-β1表达的影响

目的:探讨洛伐他汀(lovastatin)对大鼠角膜新生血管(cor-neal neovascularization,CNV)血管内皮生长因子(vascular endothelial growth factor,VEGF)和转化生长因子-β1(trans-forming growth factor-beta1,TGF-β1)表达的影响。方法:建立碱烧伤诱导的大鼠角膜新生血管的动物模型,治疗组和对照组分别给予10-5mol/L洛伐他汀溶液和生理盐水点眼,采用裂隙灯观察新生血管的生长情况,用免疫组织化学方法检测VEGF及TGF-β1的表达,并在基因水平下采用RT-PCR方法检测大鼠角膜VEGF mRNA的表达水平。结果:各时间点角膜新生血管的面积,治疗组均低于对照组,差异有显著性(P<0.05),免疫组化染色显示,治疗组VEGF及TGF-β1的表达量较对照组明显减少,在造模后4d角膜组织中VEGF mRNA的相对表达量,治疗组低于对照组(0.422±0.083vs0.786±0.126,P<0.05)。结论:洛伐他汀通过下调VEGF及TGF-β1的表达,抑制血管内皮细胞增殖,最终可有效抑制碱烧伤诱导的大鼠角膜新生血管形成。     

Placental growth factor,a member of the VEGF family,contributes to the development of choroidal neovascularization

PURPOSE: VEGF has been shown to be necessary, but not sufficient alone, for the development of subretinal pathologic angiogenesis. In the current study, the influence of placental growth factor (PlGF), a member of the VEGF family, in human and experimental choroidal neovascularization (CNV) was investigated. METHODS: The presence of VEGF family member mRNA was evaluated by RT-PCR in neovascular membranes extracted during surgery. The spatial and temporal pattern of VEGF isoforms and PlGF mRNA expression were explored by using the laser capture catapulting technique and RT-PCR in a murine laser-induced model and in vitro. PlGF expression was also studied in human donor eyes. The influence of endogenous PlGF was evaluated in deficient mice (PlGF(-/-)) and by antibody-mediated neutralization of the PlGF receptor. RESULTS: Human neovascular membranes consistently expressed VEGF-A, -B, and -C; PlGF; and VEGFR-1 and -2. The VEGF(120) isoform mRNA was primarily induced in early stages of angiogenesis in vivo and in vitro. PlGF mRNA expression was present in the intact choroid and significantly upregulated during the course of experimental CNV. Both deficient PlGF expression in PlGF(-/-) mice and PlGF receptor neutralization in wild-type mice prevented the development of choroidal neovascularization induced by laser. CONCLUSIONS: These observations demonstrate the participation of PlGF in experimental CNV. They identify therefore PlGF as an additional promising target for ocular antiangiogenic strategies.