CCR7/p-ERK1/2/VEGF signaling promotes retinal neovascularization in a mouse model of oxygen-induced retinopathy

AIM: To investigate the role of CCR7/p-ERK1/2/VEGF signaling in the mouse model of oxygen-induced retinopathy (OIR). METHODS: Neonatal C57BL/6J mice were evenly randomized into four groups: normoxia, OIR, OIR control (treated with scramble siRNA), and OIR treated (treated with CCR7 siRNA). Normoxia group was not specially handled. Postnatal day 7 (P7) mice in the OIR group were exposed to 75%±5% oxygen for 5d (P7-P12) and then maintained under normoxic conditions for 5d (P12-P17). Mice in the OIR control and OIR treated groups were given injections of scramble or CCR7 siRNA plasmid on P12 before returning to normoxic conditions for 5d (P12-P17). Retina samples were collected from all mice on P17, stained with adenosine diphosphatase (ADPase), and retinal neovascularization (RNV) was assessed. Retinas were also stained with hematoxylin and eosin (H&E) for RNV quantitation. The distribution and expression of CCR7, p-ERK1/2 and vascular endothelial growth factor (VEGF) were assessed via immunohistochemistry, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: High oxygen promoted retinal neovascularization (P<0.05) and increased the number of endothelial nuclei in new vessels extending from the retina to the vitreous body; CCR7 promoted this process (P<0.05). CCR7 and VEGF mRNA were expressed at higher levels in the OIR and OIR control groups than in the normoxia and OIR treated groups. CCR7, p-ERK1/2, and VEGF protein were expressed in the retinas of mice in the OIR and OIR control groups. Intravitreal injection of CCR7 siRNA significantly reduced CCR7, p-ERK1/2, and VEGF expression in the OIR mouse model (all P<0.05). CCR7 significantly enhanced the neovascularization and non-perfusion areas in the OIR group (P<0.05). CCR7 siRNA significantly reduced levels of p-ERK1/2 and VEGF as compared to OIR controls (P<0.05). CONCLUSION: These results suggest that CCR7/p-ERK 1/2/VEGF signaling plays an important role in OIR. CCR7 may be a potential target for the prevention and treatment of retinopathy of prematurity.     

Localization of adenosine A2a receptor in retinal development and oxygen-induced retinopathy

PURPOSE: To investigate the association of adenosine A2a receptors (A2aR) with retinal vasculogenesis and angiogenesis that occurs in the canine model of oxygen-induced retinopathy (OIR). METHODS: One-day-old dogs were exposed to 100/o oxygen for 4 days and killed in oxygen (5 days old) and at 3, 10, 17, and 23 days after exposure to hyperoxia. Room air control animals were killed at 1, 5, 8, 15, 22, and 28 days of age. Immunolocalization of A2aR was performed on frozen sections, and reaction product density was quantified using microdensitometry. Cell types were identified in serial sections using antibodies against von Willebrand factor (endothelial cells) and GFAP (astrocytes), and enzyme histochemistry for menadione-dependent a-glycerophosphate dehydrogenase (M-a-GPDH) (to label angioblasts and developing blood vessels). RESULTS: A2aR immunoreactivity was associated with forming blood vessels and angioblasts in the nerve fiber layer (NFL) of peripheral retina. As development progressed, vascular labeling decreased, whereas labeling of neuronal elements increased. In OIR, A2aR immunoreactivity in the NFL was reduced after exposure to hyperoxia and significantly elevated in the inner retina throughout vascularized retina and in advance of forming vasculature in all oxygen-treated animals returned to room air. A2aR immunoreactivity was also prominent in fronds of intravitreal neovascularization. CONCLUSIONS: A2aR immunoreactivity was associated with developing retinal vessels. As development progressed, vascular-associated A2aR labeling decreased and, concomitantly, labeling of neuronal elements increased. A2aR immunoreactivity was significantly elevated at the edge of forming vasculature in all animals returned to room air after hyperoxia and in intravitreal neovas cular formations. These results provide additional evidence for the importance of A2aR and its ligand adenosine in retinal vascular development and in the vasoproliferative stage of canine OIR.     

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。     

血管内皮生长因子反义寡聚脱氧核苷酸对增生性视网膜病变幼鼠血管内皮生长因子表达的影响

目的 观察血管内皮生长因子（vascular endothelial cell growth factor,VEGF）反义寡聚脱氧核苷酸（antisense oligodeoxynucleotides，ASODNs）对增生性视网膜病变幼鼠模型VEGF表达的影响。 方法 将30只Sprague-Dawley(SD) 新生幼鼠随机分为正常对照组、用药组及未用药组,每组10只。用高浓度氧诱导用药组及未用药组SD幼鼠，建立增生性视网膜病变幼鼠模型。对用药组幼鼠行VEGF ASODNs 球后注射，3d后取各组幼鼠除角膜、巩膜及晶状体外的全部眼内组织及血清，通过竞争性酶免疫测定法检测其VEGF的含量。 结果 用药组幼鼠眼内组织VEGF含量明显低于未用药组（P＜0.05），而与对照组相比差异无显著性的意义（P＞0.05）；用药组幼鼠血清VEGF含量与未用药组相比差异无显著性的意义（P＞0.05），两者均明显低于对照组（P＜0.05）。 结论 VEGF ASODNs能明显抑制由高浓度氧诱导的增生性视网膜病变幼鼠VEGF的表达。 （中华眼底病杂志，2003，19：172-174）     

氧诱导小鼠视网膜组织VEGF和iNOS的早期表达及意义

目的明确血管内皮生长因子（VEGF）和诱导型一氧化氮合酶（iNOS）在氧诱导小鼠视网膜组织中早期的表达特征,并探讨上述促血管生成因子的表达在缺氧状态下视网膜新生血管生成中的意义。方法新生C57BL/6J小鼠暴露于高氧环境5d后转置于相对低氧环境,以建立视网膜新生血管小鼠模型,分别于暴露低氧环境0、2、6、24h后摘除眼球,应用RT-PCR及免疫组织化学技术检测全视网膜组织中VEGF及iNOS的表达情况。结果在低氧环境下,VEGF的表达水平呈现较明显的时间依赖性特征,与0h时间点相比,缺氧2hVEGFmRNA表达升高,6h尤为明显,24h下降。iNOSmRNA表达亦呈相似的特征,其表达高峰出现在缺氧2h,6h起即呈逐渐下降趋势。VEGF主要定位于视网膜节细胞层（RGCL）及内网层（IPL）的细胞浆,而iNOS蛋白表达于GCL的细胞浆;上述蛋白表达亦呈现与基因转录水平相一致的时间依赖性。结论暴露于缺氧环境下24h内视网膜VEGF和iNOS表达均上调,且随暴露时间呈动态变化,提示视网膜缺氧性病变早期即出现促血管生成因子的表达变化。     

Immunolocalisation of the VEGF receptors FLT-1, KDR, and FLT-4 in diabetic retinopathy

AIM: To determine the spatial and temporal changes in the staining pattern of the VEGF receptors FLT-1, KDR, and the putative receptor FLT-4 during the pathogenesis of diabetic retinopathy. METHODS: Immunohistochemical localisation of VEGF receptors, using antibodies against FLT-1, FLT-4, and KDR, was carried out on specimens of normal human retina (n = 10), diabetic retinas (a) with no overt retinopathy (n = 12), (b) with intraretinal vascular abnormalities but no proliferative retinopathy (n = 5), (c) with active proliferative retinopathy (n = 6), and (d) with no residual proliferative retinopathy after scatter photocoagulation therapy (n = 14), and surgically excised diabetic fibrovascular membranes (n = 11). The degree and pattern of immunostaining was recorded. RESULTS: FLT-1 staining was apparent in the retinas from both non-diabetic and diabetic retinas; weak to moderate staining was generally confined to the inner nuclear layer, the ganglion cell layer, and the retinal vessels during all stages of the disease process. Staining of the retinal vessels was raised in diabetic tissue compared with non-diabetic tissue. The preretinal vessels of the diabetic subjects stained moderately to intensely for FLT-1. In contrast with FLT-1 staining minimal immunostaining for KDR was demonstrated in the non-diabetic eyes and the unlasered eyes; however, weak staining for KDR was observed in the inner nuclear layer and the ganglion cell layer of the unlasered eyes with diabetic changes. In those retinas with preretinal neovascularisation KDR immunoreactivity was moderate to intense in the intra- and preretinal vessels. However, in the excised membranes, where the vessels may have been in a quiescent state, the levels of KDR were weak to moderate. After apparently successful laser treatment KDR staining was reduced in the intraretinal vessels. Minimal FLT-4 staining was observed throughout normal eyes while weak to moderate FLT-4 staining was generally confined to the inner nuclear layer and the ganglion cell layer of the unlasered diabetic eyes. Weak to moderate levels of FLT-4 staining were observed in the intraretinal vessels except after apparently successful laser treatment where reduced levels of staining were observed. Weak to moderate staining was observed in the preretinal vessels. CONCLUSIONS: This study supports a role for FLT-1, KDR, and possibly FLT-4 in the pathogenesis of diabetic retinopathy; however, their specific roles in the progression of the disease may differ.     

白藜芦醇对氧诱导鼠视网膜B细胞白血病蛋白2和血管内皮生长因子表达的影响

目的探讨白藜芦醇对高氧诱导的早产儿视网膜病变新生鼠视网膜B细胞白血病蛋白2（B-cell leukemia/lymphoma-2,bcl-2）和血管内皮生长因子（vascular endothelial growth factor,VEGF）表达的影响。方法选取鼠龄为7d的Sprague-Dawley（SD）幼鼠60只,分A组（正常对照组）、B组（给氧组）和C（实验组1）、D（实验组2）、E（实验组3）三个实验组,每组12只。A组幼鼠生活在正常氧环境中,B组、C组、D组和E组幼鼠置于氧分压为（75±2）%的氧环境中连续生活5d,建立高氧诱导的早产儿视网膜病变动物模型。C、D、E三个实验组分别予白藜芦醇10mg/（kg·d）、30mg/（kg·d）、60mg/（kg·d）灌胃给药,每天一次,连续5d,所有幼鼠均于17d鼠龄处死。应用Western-blot免疫印迹法分析检测鼠视网膜bcl-2的表达,并以计算机图像分析仪进行分析;用ELISA方法检测鼠视网膜VEGF的表达。结果与A组（正常对照组）比较,B组（给氧组）视网膜bcl-2和VEGF的表达是A组的4倍,两组差异有统计学意义（P＆lt;0.01）;与B组比较,C、D、E三个实验组视网膜bcl-2和VEGF的表达逐渐减弱,呈剂量依赖关系,不同浓度的白藜芦醇实验组之间存在显著性的差异（P〈0.01）,三种不同浓度（10mg/kg、30mg/kg、60mg/kg）的白藜芦醇均显著抑制视网膜bcl-2和VEGF的表达,其抑制率分别为11.09%、38.05%、69.76%和3.42%、23.04%、43.69%。结论白藜芦醇可显著抑制高氧诱导的早产儿视网膜病变新生鼠视网膜bcl-2和VEGF表达,可能对早产儿视网膜病变等视网膜新生血管疾病具有治疗作用。     

VEGFR-1和VEGFR-2在氧诱导视网膜病变小鼠视网膜中表达的变化

背景 早产儿视网膜病变(ROP)是氧动力学异常导致严重视网膜血管病变的致盲眼病.血管内皮生长因子(VEGF)在ROP发病机制中发挥重要作用,但VEGF受体(VEGFR)在ROP发病中的作用研究较少. 目的 研究不同鼠龄氧诱导视网膜病变(OIR)小鼠视网膜中VEGFR-1和VEGFR-2的表达变化.方法 将60只SPF级出生后7 d(P7)的C57BL/6J小鼠与哺乳母鼠一起在体积分数(75±2)％O2条件下饲养5d,建立OIR小鼠模型(OIR组),以60只正常环境饲养的同品系同龄小鼠作为正常对照组.分别于P8、P11、P12、P13、P14、P18鼠龄时摘取2个组小鼠双侧眼球,制备视网膜切片,采用苏木精-伊红染色法检测小鼠视网膜血管的发育情况;采用实时荧光定量PCR法检测不同鼠龄小鼠视网膜中VEGFR-1和VEGFR-2 mRNA的相对表达水平;采用免疫组织化学法观察小鼠视网膜中VEGFR-1和VEGFR-2蛋白的表达.结果 组织病理学结果显示,OIR组P13、P14、P18小鼠有较多的血管内皮细胞核突破内界膜,光学显微镜下内界膜下的细胞增生,排列紊乱,视网膜表面或视网膜前有新生血管芽,但正常对照组各鼠龄小鼠视网膜结构正常.OIR组P12、P13、P14和P18小鼠视网膜中VEGFR-1 mRNA的平均相对表达水明显高于正常对照组,差异均有统计学意义(P=0.046、0.000、0.000、0.042);OIR组P8、P11、P12、P13、P14和P18小鼠视网膜中VEGFR-2mRNA的平均相对表达水平明显高于正常对照组,差异均有统计学意义(均P＜O.01).两个组间P8、P11、P12、P13、P14、P18小鼠视网膜中VEGFR-1蛋白阳性表达强度的差异均无统计学意义(M=50.00、36.00、41.00、31.00、28.00、36.00,均P＞0.05);正常对照组与OIR组间P8和P11小鼠视网膜中VEGFR-2蛋白阳性反应强度的差异无统计学意义(均P＞0.05),正常对照组P12、P13小鼠视网膜中VEGFR-2蛋白的表达减弱,但OIR组同鼠龄小鼠视网膜中VEGFR-2蛋白的表达增强,2组间差异均有统计学意义(均P＜0.01);2个组P14小鼠视网膜中VEGFR-2蛋白表达均明显增强,但OIR组的表达强度仍显著高于正常对照组,差异有统计学意义(P＜0.01).正常对照组P18小鼠视网膜中VEGFR-2蛋白表达减弱,OIR组VEGFR-2表达强度达峰(M=20.11,P＜0.01). 结论 VEGFR参与OIR小鼠视网膜新生血管的形成,VEGFR-2的促新生血管新生作用强于VEGFR-1.     

Expression of pro- and anti-angiogenic isoforms of VEGF in the mouse model of oxygen-induced retinopathy

Retinopathy of prematurity (ROP) has become one of the leading causes of blindness and visual loss in children over the last half century. Vascular Endothelial growth Factor (VEGF-A) is the principal stimulator of angiogenesis. Recently, it has been identified that VEGF was differentially spliced from Exons 8 to Exons 8a and 8b to form two families: the pro-angiogenic VEGFxxx family and the anti-angiogenic VEGFxxxb family. This alternate splicing produced VEGFxxxb proteins of the same length as VEGFxxx family, but with different C terminal amino acid sequences. VEGFxxxb appeared to be able to inhibit VEGFxxx-dependent angiogenesis. In our study, we investigated the protein expression course of VEGFxxx and VEGFxxxb by Western-blot in a mouse model of Oxygen-induced Retinopathy (OIR) from postnatal day 1 (P1) to postnatal day 21 (P21). We also analyzed the relative protein expression level of VEGF₁₆₅b isoform in the OIR mouse model. We found that both VEGFxxx and VEGFxxxb were present in the mouse retina, among which, VEGF₁₆₄ and VEGF₁₆₅b appeared to be predominant VEGFxxx and VEGFxxxb isoforms respectively in the mouse retina. We also found that the two family had different expression pattern correlated with neovascularization development and that the relative expression level of VEGF₁₆₅b isoform switched during the neovascularization development in the OIR mouse model. In OIR group, the protein level of total VEGF isoforms (a mix of VEGF₁₆₄ and VEGF₁₆₅b, detected by pan-VEGF antibody) continuously increased and peaked at P17 while VEGF₁₆₅b continuously decreased from P9 which was well related with the vessel obliteration and neovascularization development in the mouse model of OIR. The neovascularization development correlates with an increase of total VEGF isoforms and the decrease of VEGF₁₆₅b, indicating that there is a pro-angiogenic VEGF shift. Therefore, anti-angiogenic therapy that could alter the ratio of VEGFxxxb/VEGFxxx may be more effective.     

Effect of VEGF trap on normal retinal vascular development and oxygen-induced retinopathy in the dog

Purpose. To evaluate the effects of a vascular endothelial growth factor trap (VEGF Trap) on retinal vascular development and pathologic neovascularization (NV) in the canine model of oxygen-induced retinopathy (OIR). Methods. Newborn dogs (postnatal day [P]1) were exposed to 100% O(2) and then returned to room air on P5. VEGF Trap (5, 25, or 250 μg) was injected intravitreally in one eye and human FC (hFc) injected in the fellow eye of air control and oxygen-treated dogs on P8. The retinal vasculature and NV were evaluated on P21. Other oxygen-exposed animals received 5 μg of VEGF Trap or hFc on P22 after confirmation of retinopathy of prematurity (ROP)-like pathology and were evaluated at P45. Results. In air controls, both the vascularized area of the retina and the density of superficial capillaries were reduced in 250 or 25 μg VEGF Trap-injected eyes, and deep capillaries were absent. Eyes that received the 5 μg dose were indistinguishable from controls. In oxygen-treated animals, all eyes injected with VEGF Trap exhibited markedly less intravitreal NV than that of hFc-injected fellow eyes, irrespective of dose. Retinal vascular area in OIR animals was significantly reduced in eyes injected with 250 or 25 μg of VEGF Trap, but the 5 μg dose did not inhibit retinal revascularization. Eyes with existing NV that received 5 μg VEGF Trap at P22 exhibited substantial resolution of OIR pathology at P45. Conclusions. The VEGF Trap inhibited the formation of NV, but higher doses also inhibited revascularization of retina when injected at P8. In contrast, the lowest dose tested effectively blocked NV and caused regression of existing NV, without appreciably affecting vasculogenesis or retinal revascularization. These findings suggest that dose selection is an important variable when considering the use of VEGF-targeting agents for the treatment of ROP.     

Investigating the effects of carvacrol in rats using oxygen-induced retinopathy model

Purpose:Investigating the effects of intraperitoneal carvacrol administration in rats using the oxygen-induced retinopathy (OIR) model.Methods:A total of 28 newborn Sprague Dawley rats were used and the OIR model was created using the 50/10% oxygen model. The study composed of four groups in total. While the OIR model was not used in Group I (control group), it was created for Groups II, III, and IV. About 0.01 mL carvacrol, bevacizumab, or 0.9% NaCl was administered intraperitoneal (IP) to the rats in all groups on postnatal day (PND) 14 as follows: Group I and Group II were administered 0.9% NaCl, Group III was administered bevacizumab, and Group IV was administered carvacrol. On PND 18, rats were sacrificed and their right eyes were enucleated.Results:Histopathological and immunohistochemical studies showed that the number of vascular endothelial cells (VECs), vascular endothelial growth factor (VEGF), and tumor necrosis factor-α (TNF-α) decreased similarly in Group III and Group IV compared with Group II. VECs values for Group I, Group II, Group III, and Group IV were measured as 0 ± 0, 26.45 ± 4.57, 7.75 ± 1.98, and 5.78 ± 1.72, respectively, and it differed significantly between groups (P < 0.001). Likewise, VEGF levels were observed as 0.06 ± 0.01, 3.31 ± 0.53, 2.47 ± 0.44, and 2.49 ± 0.52, respectively, and it differed significantly between groups (P < 0.001). TNF-α levels were recorded as 0.06 ± 0.01, 3.58 ± 0.38, 2.46 ± 0.49, and 2.29 ± 0.25, respectively, and it differed significantly between groups (P < 0.001). VECs, VEGF, and TNF-α were similar between Group III and IV (range of P values were 0.486–0.998).Conclusion:The study demonstrated that carvacrol significantly reduced retinal pathological angiogenesis, NV, VEC nuclei count, VEGF, and TNF-α levels. Moreover, the observed effects were comparable to those of bevacizumab.     

Kinetics of retinal vaso-obliteration and neovascularisation in the oxygen-induced retinopathy (OIR) mouse model

Purpose  To evaluate the kinetics of peripheral vascularisation, central vessel regression and neovascularisation in the OIR mouse model in order to: i) generate standard kinetics for further studies in this model, and ii) define optimal time points to investigate cellular mechanisms of retinal vascular plasticity. Methods  From postnatal day 7 (P7) until P12, newborn mice were kept at 75% oxygen. The animals were sacrificed on different time points, during and after O₂ exposure. After intracardial perfusion with FITC–dextran, retinal flatmounts were prepared, and the size of the retinal vascular network, the size of the central avascular area, and the number of blood vessel tufts and clusters were determined. In addition to the fluorescein stain for perfused capillaries, endothelial cells were stained with isolectin. Results  Upon O₂ exposure, there is a rapid depletion of capillaries starting adjacent to the large central arteries. These avascular stripes fuse to form an avascular central area which amounts to 37% of the whole retinal surface after 2 days of hyperoxia. The peripheral capillary network remains intact throughout the incubation period, even though the pace of its centrifugal spreading is decelerated compared to room air controls. Already during O₂ exposure, revascularisation of the central avascular area is initiated by peripheral vessels sprouting in a centripetal direction. Revascularisation is accelerated after the return to room air, and is completed at P25. Maximal pathological neovascularisation can be found at P17, at the border between the avascular and vascular retina. Conclusion  Hyperoxia leads to a rapid development of a central avascular area of the retina, with its maximum during not at the end of the hyperoxic phase. Central capillary loss and peripheral vascularisation take place simultaneously, indicating different cellular control mechanisms for different areas of the retina. These standard kinetics for peripheral vascularisation and central vessel regression will: 1) help to compare the effects of angio-modulation, and 2) serve as normal baseline for the characterization of knock-out mice strains with regard to gene-specific vascular changes in the OIR-model. None of the authors has financial relationships with companies or organisations mentioned in the study     

内皮抑素抑制氧致视网膜病变小鼠 新生血管形成的实验研究

目的观察血管生成抑制因子内皮抑素（ES）对视网膜新生血管形成的抑制作用。方法将鼠龄为7 d的32只C57BL/6J小鼠随机分为给氧组（12只）、ES治疗组（12只）和对照组（8只）。将给氧组和ES治疗组小鼠置于浓度为（75±5）％高氧环境中生活5 d，然后回到正常氧环境中。ES治疗组小鼠在出氧箱后12、36 h，一只眼玻璃体腔内注射1μgES，另一只眼注射1 μl的磷酸盐缓冲溶液（PBS）作为对照。对照组小鼠生活在正常氧环境中。右旋糖苷-异硫氰酸荧光素（FITC-dextran）视网膜造影整装铺片了解视网膜新生血管改变；计数突破内界膜的内皮细胞数反映视网膜血管增生情况，观察ES对视网膜新生血管形成的抑制作用。结果与给氧组相比，ES治疗组视网膜铺片见视网膜血管分支走行正常，未见明显的无灌注区；ES治疗眼平均每个视网膜切面可见突破内界膜的内皮细胞核数减少，为（5.39±1.52）个，与给氧组［（22.56±2.13）个］比较，差异有统计学意义（P<0.001）。结论ES可有效抑制视网膜新生血管的形成，有望成为治疗血管增生性视网膜病变的新途径。(中华眼底病杂志,2005,21:314-317)     

Arresten在氧诱导小鼠视网膜新生血管形成中的抑制作用及机制

目的　探讨Arresten在氧诱导小鼠视网膜新生血管形成中的抑制作用及机制。方法　选取48只出生后7 d健康清洁级C57BL/6J幼鼠,随机分为氧诱导视网膜病变（oxygen induced retinopathy,OIR）组、OIR+ Arresten组和常氧组,每组16只。OIR+Arresten组及OIR组幼鼠在体积分数为（75±2）％的高氧环境下饲养5 d,然后转移至正常氧气环境中饲养5 d,其中OIR+Arresten组幼鼠于高氧饲养刚结束时,给予双眼玻璃体内注射Arresten蛋白。常氧组幼鼠则在常规氧气环境中连续饲养10 d。在鼠龄17 d时,各组取6只幼鼠经股静脉注射异硫氰酸葡聚糖溶液处死并摘出眼球,视网膜铺片后观察视网膜的血管形态、计算无灌注区面积。同时对小鼠眼球视网膜行石蜡切片和HE染色,计算侵入玻璃体内血管内皮细胞核的数量。同时,通过细胞培养实验,利用MTT法检测Arresten蛋白对人血管内皮细胞增殖的抑制作用。结果　视网膜铺片结果显示,常氧组、OIR组、OIR+Arresten组视网膜无灌注区相对面积分别为（2.35±1.62）％、（57.28±9.36）％和（20.38±8.69）％,三组间总体比较,差异有统计学意义（F=18.732,P<0.05）,且OIR组无灌注区相对面积显著高于OIR+Arresten组,差异有统计学意义（P<0.05）。常氧组小鼠视网膜的内界膜结构仍保持平滑完整,未发现有新生血管侵入玻璃体内。OIR组和OIR+Arresten组每张切片上血管内皮细胞核的数量分别为 （15.18±4.83）个、（7.33±3.88）个,其中OIR+Arresten组侵入玻璃体内新生血管内皮细胞核的数量显著低于OIR组,差异有统计学意义（P<0.05）。Arresten蛋白对人脐静脉内皮细胞增殖的抑制率随着其浓度的升高而增加,但当Arresten蛋白浓度达到1000 μg·L^-1时,人脐静脉内皮细胞的增殖抑制率达到顶峰,为（58.00±0.65）％。结论　Arresten蛋白能够抑制氧诱导的小鼠视网膜新生血管形成,通过抑制血管内皮细胞增殖来抑制新生血管形成。     

Cyclin D1在氧诱导视网膜病变大鼠视网膜中的表达

目的研究氧诱导视网膜病变(oxygen-induced retinopathy,OIR)中cyclin D1表达的变化对视网膜新生血管增殖的影响。方法 120只鼠龄7d SD大鼠随机分为实验组和对照组2组。实验组60只大鼠置于体积分数75%高氧环境5d建立OIR模型,对照组60只大鼠置于正常空气中。2组分别于生后第12天、第14天、第17天、第22天、第26天各取12只大鼠并均取双侧眼球,左眼做视网膜铺片ADP酶染色观察视网膜新生血管,免疫组化及免疫荧光观察cyclin D1的表达。右眼视网膜行Western blot检测cyclin D1蛋白表达情况。结果视网膜铺片ADP酶染色观察到大鼠生后第14天开始出现新生血管,至第17天新生血管反应最大,随后新生血管缓慢退化,在生后第26天血管结构趋于正常。免疫组化和免疫荧光显示大鼠生后第14天、第17天、第22天实验组视网膜cyclin D1呈高表达。Western blot结果显示cyclin D1蛋白的表达于生后第12天比对照组低,第14天开始增高,第17天达到最高,第26天降低,但高于对照组。Cyclin D1表达的变化符合新生血管增殖的过程。结论 cyclin D1的异常表达可能与视网膜新生血管增殖密切相关。     

共培养系统中视网膜微血管周细胞经KDR/Flk-1途径对内皮细胞增生的影响

目的:采用细胞共培养模型研究缺氧诱导视网膜新生血管生成过程中周细胞对微血管内皮细胞增生的作用及其相关血管内皮细胞生长因子受体2(KDR/Flk-1)调节机制。方法:免疫磁珠法原代分离培养大鼠视网膜微血管内皮细胞和周细胞,分别采用抗VIII因子、CD31抗体以及抗血小板源性生长因子受体β、结蛋白抗体免疫细胞化学法鉴定内皮细胞和周细胞。通过Millicell小室建立周细胞和内皮细胞共培养模型,使用MTT法和流式细胞仪检测内皮细胞增殖数量及周期,观察缺氧和常氧下周细胞对血管内皮细胞增生的影响,并采用RT-PCR法检测内皮细胞KDR/Flk-1的mRNA水平变化,探讨相关调节机制。结果:经免疫细胞化学法鉴定,免疫磁珠法可获得高纯度的视网膜微血管内皮细胞和周细胞。MTT法显示,缺氧3～9d内皮细胞增生明显,6d增生达高峰(24.9%,P<0.01);共培养时内皮细胞的增生可被周细胞抑制。流式细胞仪检测发现,缺氧6dS期内皮细胞数量明显增加(43.9%,P<0.01);常氧(3.6%,P<0.05)或缺氧(15.1%,P<0.01)共培养时,周细胞均可抑制内皮细胞增生。缺氧下内皮细胞KDR/Flk-1的mRNA水平是常氧下的1.3倍;常氧(45.1%,P<0.05)和缺氧(27.7%,P<0.05)共培养下,周细胞均可下调内皮细胞KDR/Flk-1的mRNA表达。结论:缺氧和常氧共培养时周细胞均可抑制微血管内皮细胞增生,此抑制作用部分是通过下调内皮细胞KDR/Flk-1mRNA表达实现。     

Inhibition of proliferation of retinal microvascular endothelial cells by pericytes through down-regulating KDR/Flk-1 in a co-culture system

To investigate the role of pericytes in growth of retinal microvascular endothelial cells(RMECs) in a co-culture system in order to understand some mechanism of angiogenesis in hypoxia induced retinal neovascular disorders. · METHODS: RMECs were isolated by a modified protocol using CD31 coated Dynabeads, and identified by immunocytochemical staining with anti-Factor VIII and CD31 antibodies. Rat retinal pericytes were isolated and characterized by immunofluorescent staining with PDGFR-β; and desmin antibodies. Pericytes and RMECs were cultured in a contact co-culture system both under normoxia and hypoxia by Millicell chamber. RMECs proliferation was evaluated by MTT and cell cycle assay with flow cytometry. RT-PCR was used to detect the alteration of KDR/Flk-1 mRNA level in RMECs under normoxia or hypoxia in the co-culture system. · RESULTS: Highly pured rat RMECs and pericytes were harvested with the modified isolating method. The two cell types were identified by positive Factor VIII, CD31 and PDGFR-β, desmin cytochemical staining respectively. RMECs proliferated significantly under hypoxia from 3 to 9 day with a maximal rate on day 6 (24.9%, P < 0.01) by MTT. In the co-culture system, the proliferation of RMECs was inhibited by pericytes. After 6 days exposure to hypoxia, the fraction of S-phase RMECs number was greatly increased by 43.9%(P < 0.01). In the co-culture system, RMECs proliferation was inhibited by pericytes through decreasing the fraction of S-phase cell number both under normoxia (3.6%, P <0.05) and under hypoxia (15.1%,P <0.01). KDR/Flk-1 mRNA level in single cultured RMECs was shown to increase approximately 1.3-fold when exposed to hypoxia. Compared with single cultured RMECs, co-culture with pericytes could decrease KDR/Flk-1 mRNA by 45.1% (P <0.05) and 27.7% (P <0.05) under normoxia and hypoxia condition respectively. · CONCLUSION: The present study demonstrated that pericytes could inhibit proliferation of RMECs under both normoxia and hypoxia. The inhibition effects of pericytes maybe, at least in part, due to downregulation of KDR/Flk-1 of RMECs. These findings confirm that pericytes could be a potential inhibitor in the pathogenesis of retinal neovascularization(RNV).     

Colocalization of neuropilin-1 and Flk-1 in retinal neovascularization in a mouse model of retinopathy

PURPOSE. To investigate the mechanisms of the development of retinal neovascularization, the localizations of vascular endothelial (VEGF) receptors Flk-1 and neuropilin (NP)-1 mRNAs were examined. METHODS. The model of retinopathy of prematurity (ROP) was produced by ischemia-induced ocular neovascularization, by exposing postnatal day-7 mice to 75% oxygen for 5 days and then returning them to room air for 5 days. Retinal neovascularization was visualized by injection of fluorescein-dextran. Expression of Flk-1 and NP-1 mRNAs were examined by in situ hybridization with flatmount and serial sections of the retina. The localization of NP-1 was also confirmed by immunohistochemistry. Blood vessel patterns were characterized by immunohistochemical localization of von Willebrand factor (vWF). RESULTS. Flatmount in situ hybridization showed intense expression of NP-1 and Flk-1 mRNAs colocalized in the area of neovascularization. In situ hybridization of serial sections of the retina revealed that expression of Flk-1 and NP-1 was restricted to neovascularized vessels of the retina from ROP mice. CONCLUSIONS. The restricted expression of Flk-1 and NP-1 on neovascularized vessels suggests that these molecules may play important roles in retinal neovascularization. This is the first report of the colocalization of NP-1 and Flk-1 on neovascularized vessels of the retina from ROP mice.