靶向血管内皮生长因子治疗眼新生血管的研究进展

眼新生血管的形成严重破坏眼的结构和功能,引起不同程度的视力障碍,因此研究新生血管的生成机制并寻找有效的治疗方案是目前急需攻克的研究课题.血管内皮生长因子(vascular endothelial growth factor,VEGF)是目前发现的最为强大和专一的刺激内皮细胞增生的因子,在新生血管生成中起着中心作用.通过眼内注射VEGF 受体嵌合蛋白、抗VEGF单克隆抗体、靶向VEGF 小干扰RNA均可不同程度地抑制眼内新生血管生成.本文就靶向VEGF治疗眼新生血管的研究进展进行综述.     

血管内皮生长因子与眼内新生血管

眼内新生血管是多种致盲眼病的病理生理基础,新生血管形成受多种因子调控,其中血管内皮生长因子(VEGF)是最重要的细胞因子。它与特异性受体结合后通过复杂机制促进血管内皮细胞增殖、迁移和通透性增加。缺氧可诱导VEGF表达,VEGF通过NO的介导作用发挥促血管新生的作用。还有多种抑制和促进血管因子共同作用于血管内皮细胞。目前,人们通过基因手段抑制VEGF及其受体达到抑制血管新生的作用,为眼内新生血管性疾病开辟前景。     

VEGF与其他DR相关细胞因子关系的研究进展

血管内皮生长因子(vascular endothelial growth factor,VEGF)是目前发现与糖尿病视网膜病变(diabetic retinopathy,DR)中新生血管形成联系最密切的细胞因子之一。抗VEGF药物是目前最有效的抑制DR新生血管形成的手段,除此之外尚无其他有效治疗。现发现部分DR相关细胞因子与VEGF有关,可能在DR新生血管过程中发挥作用。本文就VEGF与其他DR相关细胞因子的关系做一综述。     

血管内皮生长因子抑制剂治疗视网膜新生血管性疾病

视网膜新生血管性疾病是致盲的主要病因,早期预防、控制视网膜新生血管的发生发展显得尤为重要。目前,关于视网膜新生血管性疾病的确切发病机制尚不清楚,但对血管内皮生长因子(vascular endothelial growth factor,VEGF)在其形成过程中起关键作用已达成共识,现在已有多种VEGF抑制剂被用于治疗眼部新生血管性疾病。现将治疗视网膜新生血管性疾病的最新几种VEGF抑制剂作一综述。     

视网膜血管性疾病患者玻璃体液中血管内皮生长因子和肿瘤坏死因子-α的含量测定

新生血管形成导致的玻璃体积血、牵引性视网膜脱离是引起增生型糖尿病视网膜病变(PDR)、视网膜分支静脉阻塞(BRVO)等视网膜血管性疾病患者视力丧失的主要原因[1].血管内皮生长因子(VEGF)是目前已知最强的致血管通透因子,临床上应用抗VEGF药物治疗新生血管形成取得了一定疗效[2.但新生血管形成受体内多种细胞因子的调控,单纯抗VEGF治疗并不能完全抑制病理性新生血管形成[3].     

VEGF映射ROP新生血管的初步研究

目的:利用VEGF的变化映射早产儿视网膜病变新生血管的发生发展过程。方法:建立缺氧小鼠新生血管动物模型,实时定量PCR检测小鼠视网膜VEGF在不同时间点的mRNA值,通过函数绘制VEGF与新生血管的拟合曲线,并求得VEGF映射视网膜新生血管的函数方程。结果:通过实验和数学推算,我们获得VEGF映射视网膜新生血管的线性方程。结论:利用VEGF的mRNA表达,可以建立方程映射视网膜新生血管的发生发展,为下一步研究奠定基础。     

血管内皮生长因子在氧诱导小鼠新生血管中的表达及意义

目的探讨血管内皮生长因子（VEGF）在小鼠视网膜新生血管中的表达及意义。方法对新生c57BL／6N小鼠高氧后相对低氧饲养，诱导产生视网膜新生血管。在出生后12d、17d摘除眼球，应用RT-PCR，Western Blot以及视网膜血管荧光灌注造影技术检测全视网膜VEGF mRNA、蛋白表达水平以及视网膜新生血管的发生程度。结果视网膜血管造影显示高氧造成血管发育受限，相对低氧后产生新生血管。伴随新生血管的发生，VEGF mRNA升高2．3倍；VEGF蛋白含量也升高7．3倍。结论VEGF表达改变与新生血管发生成正相关，其升高也是造成病理性视网膜新生血管发生的机制之一。减少内源性VEGF表达可能成为治疗视网膜新生血管的新方法。     

血管内皮生长因子-B与眼部新生血管性疾病

异常的新生血管会导致多种眼部疾病,而血管内皮生长因子（VEGF）在新生血管的发生及发展中起重要作用.VEGF-B作为VEGF家族的一员,对血管生成和血管通透性无显著影响,但可通过血管生存作用和细胞凋亡作用对新生血管的生长进行调控,从而抑制新生血管的发生和发展.同时,VEGF-B对心脏和神经元等具有保护作用.抗VEGF治疗作为目前明确有效的治疗新生血管的方法,一直受到广泛关注,而因VEGF-B的两面性,对于抗VEGF-B的药物在治疗眼科新生血管性疾病方面的作用尚需进一步研究.就VEGF-B对机体血管、细胞、神经元及心脏所起的作用及其在眼部新生血管性疾病治疗中的应用进行综述.     

雌激素干预早产儿视网膜病变的研究进展

早产儿视网膜病变(ROP)是一种以视网膜新生血管为主要病理改变的疾病,是儿童致盲的主要原因之一。目前主要采取激光或手术治疗,损伤较大且效果不理想,因此寻找有效的药物途径来预防ROP的发生成为研究热点。血管内皮生长因子(VEGF)是一种已知的促新生血管生成物质,在ROP的发病过程中起关键作用,雌激素通过调控VEGF减少新生血管生成,从而防止ROP的发生。本文将围绕早产儿视网膜病变新生血管,VEGF以及雌激素三者之间的关系进行综述。     

VEGF在糖尿病视网膜病变发病机制中的作用及抗VEGF靶向治疗新进展

糖尿病视网膜病变(diabetic retinopathy,DR)是糖尿病最常见的并发症之一。DR的发病机制十分复杂,至今尚未完全阐明。目前认为DR患者会出现视网膜毛细血管阻塞,可导致局部缺氧,由此引起促新生血管因子产生增多,其中包括VEGF。VEGF是促新生血管形成的主要因子之一,会降低血管内皮细胞间的紧密连接蛋白表达,参与血管炎症反应、增加血管通透性和促进新生血管形成。近年来有关DR的发病机制以及治疗的研究越来越深入。本文旨在阐述VEGF在DR发病机制中的作用,以及抗VEGF治疗策略的最新进展。     

VEGF164(165) as the pathological isoform: differential leukocyte and endothelial responses through VEGFR1 and VEGFR2

PURPOSE: Vascular endothelial growth factor (VEGF) induces angiogenesis and vascular permeability and is thought to be operative in several ocular vascular diseases. The VEGF isoforms are highly conserved among species; however, little is known about their differential biological functions in adult tissue. In the current study, the inflammatory potential of two prevalent VEGF isoform splice variants, VEGF(120(121)) and VEGF(164(165)), was studied in the transparent and avascular adult mouse cornea. METHODS: Controlled-release pellets containing equimolar amounts of VEGF(120) and VEGF(164) were implanted in corneas. The mechanisms underlying this differential response of VEGF isoforms were explored. The response of VEGF in cultured endothelial cells was determined by Western blot analysis. The response of VEGF isoforms in leukocytes was also investigated. RESULTS: VEGF(164) was found to be significantly more potent at inducing inflammation. In vivo blockade of VEGF receptor (VEGFR)-1 significantly suppressed VEGF(164)-induced corneal inflammation. In vitro, VEGF(165) more potently stimulated intracellular adhesion molecule (ICAM)-1 expression on endothelial cells, an effect that was mediated by VEGFR2. VEGF(164) was also more potent at inducing the chemotaxis of monocytes, an effect that was mediated by VEGFR1. In an immortalized human leukocyte cell line, VEGF(165) was found to induce tyrosine phosphorylation of VEGFR1 more efficiently. CONCLUSIONS: Taken together, these data identify VEGF(164(165)) as a proinflammatory isoform and identify multiple mechanisms underlying its proinflammatory biology.     

猴眼玻璃体腔内注射抗VEGF药物对视网膜色素上皮层和光感受器内节VEGF含量的影响

目的：：研究比较抗血管内皮生长因子( VEGF )药物(贝伐单抗、雷珠单抗、阿柏西晋)行猴眼玻璃体腔内注射后对视网膜色素上皮层和眼内节VEGF的作用效果。方法：选取14只健康猕猴,4只双眼注射贝伐单抗,4只双眼注射雷珠单抗,4只双眼注射阿柏西晋,每种注射药物的4只猴双眼分别于注射药物后第一天和第七天分别摘除2只猴的眼球,剩余2只未注射任何药物猴的双眼作为对照。所有摘除后的眼球福尔马林固定,石蜡包埋,切片后给予抗 VEGF 抗体,用光学显微镜观察,经 Image-Pro Plus软件处理图片,用JMP10.0进行统计学分析。结果：这三种药物均能降低视网膜色素上皮( RPE)和光感受器內节( inner segment)的VEGF水平,贝伐单抗作用在三种药物中作用最强,雷珠单抗在注射后第一天与阿柏西晋注射后第一天相比,雷珠单抗作用较强,但二者在注射后第七天,作用基本相似。结论：这三种药物均能降低RPE和inner segment的VEGF水平。     

土槿乙酸对高糖环境下人视网膜微血管内皮细胞表达血管内皮生长因子的影响

目的　观察土槿乙酸对高糖环境下人视网膜微血管内皮细胞（human retinal microvascular endothelial cells,HRMEC）表达血管内皮生长因子（vascular endothelial growth factor,VEGF）的影响。方法　体外培养HRMEC,根据不同的实验目的将其分为低糖空白对照（PL）组、土槿乙酸对照（P0）组、高糖对照（PH）组以及不同浓度土槿乙酸+高糖（P1、P2、P3）组。采用MTT法检测土槿乙酸作用后HRMEC的增殖情况,实时定量PCR和Western blot检测VEGF的mRNA和蛋白表达以及核转录因子FOXO3a的磷酸化,免疫荧光检测FOXO3a的核转位,染色质共沉淀检测FOXO3a和VEGF启动子的结合。结果　PH组HRMEC增殖率和VEGF表达水平明显高于PL组,并且随着时间的延长,HRMEC增殖率和VEGF表达均逐渐增高（均为P<0.05）;而经不同浓度土槿乙酸处理后的P1组、P2组和P3组中的HRMEC增殖率及VEGF表达水平与PH组相比,差异也均有统计学意义（均为P＜0.05）。Western blot结果显示,P0组HRMEC中FOXO3a磷酸化水平较低,PL组和PH组均有不同程度增高,而P1、P2、P3组随着土槿乙酸浓度的增加,FOXO3a磷酸化水平随之降低。免疫荧光和染色质共沉淀结果显示,P0组HRMEC中FOXO3a在细胞核内含量较多,和VEGF启动子的结合水平较高;而在PL组和PH组,细胞浆中FOXO3a明显增多,核内FOXO3a和VEGF的结合水平低于P0组;P1组、P2组及P3组中细胞核内FOXO3a以及FOXO3a-VEGF DNA复合体明显增多。结论　土槿乙酸能抑制高糖条件下HRMEC的增殖,最终激活核转录因子FOXO3a,从而下调HRMEC表达VEGF。     

缺氧对小鼠角膜血管内皮生长因子及可溶性fms-样酪氨酸激酶受体-1表达的影响

目的观察缺氧对小鼠角膜组织中血管内皮生长因子（vascular endothelial growth factor,VEGF）及可溶性fins一样酪氨酸激酶受体-1（soluble fms-like tyrosine kinase receptor-1, sFlt-1 ）表达的影响并探讨两者之间的关系。方法实验用健康雌性成年昆明小鼠70只，分为缺氧组和正常对照组，缺氧仓的氧气体积分数为8％～10％，缺氧时间分别为1、3、5、7、10、14d。用变性Western blot技术检测小鼠在缺氧条件下角膜组织中总VEGF表达的变化，用非变性Western blot技术检测小鼠在缺氧条件下角膜组织中游离及结合VEGF表达的变化，用逆转录聚合酶链反应技术检测小鼠在缺氧条件下角膜组织中VEGF、sFlt-1、缺氧诱导因子-1（hypoxia in-ducible巧玲珑factor-1，HIF-1）的基因表达变化。结果缺氧14d内未见小鼠角膜新生血管。缺氧组总VEGF及结合VEGF表达增加，随着缺氧时间的延长而增强，与正常对照组比较差异有统计学意义（P〈0.05），但缺氧14d内均未见游离VEGF表达。缺氧组VEGF、sFh-1、HIF-1的基因表达均增加，随着缺氧时间的延长而增强，与正常对照组比较差异有统计学意义（P〈0．05），其中VEGF的基因表达与HIF．1的基因表达呈正相关（r=0．993），sFlt-1的基因表达与HIF-1的基因表达呈正相关（r=0．997）。结论缺氧可以诱导小鼠角膜组织中VEGF和sFlt-1表达的增加，在缺氧的条件下，小鼠角膜组织中VEGF和sFlt-1呈平衡状态。sFlt-1可作为一种内源性VEGF拮抗因子治疗眼部新生血管性疾病。（中国眼耳鼻喉科杂志，2008，8：286-288）     

Induction of vascular endothelial growth factor after application of mechanical stress to retinal pigment epithelium of the rat in vitro

PURPOSE: To investigate the response to mechanical stress of vascular endothelial growth factor (VEGF) production by cultured retinal pigment epithelial (RPE) cells. METHODS: A pulsatile stretch device was used in vitro. RPE cells of the second passage were seeded onto flexible-bottomed culture plates; then, at subconfluent culture, the plates were subjected to pulsatile stretch. Culture plates prepared in the same way but not subjected to stretch were used as controls. After stretching for 1 hour or 24 hours, conditioned medium for measurement of VEGF production by RPE cells was collected using a mouse VEGF immunoassay. To study the expression of VEGF in RPE cells, passaged-cultured RPE cells were exposed to pulsatile stretch for 0, 1, 3, or 14 hours. Total cytoplasmic RNA was then prepared from the RPE cells. Northern blot analysis was performed for VEGF, with G3PDH used as an internal control. RESULTS: The expression and secretion of VEGF in RPE cells were increased by pulsatile stretching. CONCLUSIONS: Results indicate that stretching of the RPE could result in increased production of VEGF, with associated risk for neovascularization and changes in the blood-retinal barrier.     

反应停对大鼠碱烧伤诱导角膜新生血管的影响

目的:研究反应停(thalidomide)对碱烧伤诱导角膜新生血管(corneal neovascularization,CNV)的影响。方法:建立大鼠角膜碱烧伤诱导CNV模型,SD大鼠36只随机分成角膜新生血管对照组(A组)、反应停灌胃组(B组)和反应停球结膜下注射组(C组)。采用裂隙灯照相、免疫组化和RT-PCR等方法,检测碱烧伤后各时间点不同处理方法大鼠CNV的生长情况及VEGF的表达情况。结果:B组和C组的角膜新生血管面积和VEGF的表达均显著少于A组(P均(0.05);C组的角膜新生血管面积和VEGF的表达少于B组(P均<0.05)。结论:反应停可有效抑制大鼠角膜碱烧伤诱发CNV的生长,降低角膜新生血管VEGF的表达。球结膜下注射较全身应用更有效。     

An experimental study of VEGF induced changes in vasoactivity in pig retinal arterioles and the influence of an anti-VEGF agent

ABSTRACT: BACKGROUND: Vascular endothelial growth factor (VEGF) plays an important role in ocular physiology. Anti-VEGF agents are now used for treatment of common retinal diseases. This study characterises the vasoactive properties of VEGF in isolated perfused pig retinal arterioles under normal tone or endothelin-1 (ET-1) pre-contracted conditions and determines the influence of an anti VEGF agent on VEGF induced vasoactivity. METHODS: An isolated perfused retinal arteriole preparation was used. The outer diameter of retinal vessels was monitored at 2 second intervals in response to VEGF and the anti VEGF agent, bevacizumab. The effect of intraluminal delivery of VEGF was determined over a wide concentration range (10-16 to 10-7 M) both with and without pre-contraction with ET-1 (3 x 10-9 M). Bevacizumab (0.35 mg mL-1) was applied extraluminally to determine the influence of bevacizumab on VEGF induced vasoactive changes on ET-1 pre-contracted vessels. RESULTS: In retinal arterioles with normal tone, VEGF induced a concentration dependent contraction at low concentrations, reaching 93.5% at 10-11 M and then contraction was reduced at higher concentrations, recovering to 98.1% at 10-7 M. VEGF produced a potent concentration dependent vasodilatation in arterioles pre-contracted with ET-1. VEGF induced vasodilatation in arterioles pre-contracted with ET-1 was significantly inhibited by bevacizumab. CONCLUSIONS: VEGF induced vasoactive changes in pig retinal arterioles are dependent on concentration and vascular tone. Bevacizumab inhibits VEGF-induced vasodilatation in pre-contracted arterioles.