Involvement of purinergic P2 receptors in experimental retinal neovascularization

PURPOSE: The present study was aimed to investigate the expression of purinergic P2 receptors in oxygen-induced retinal neovascularization. METHODS: Immunohistochemistry was used to study the expression of purinergic P2Y2 and P2X2 receptors in the neonatal mouse retina during normal vascular development and after oxygen-induced retinopathy (OIR). The effect of the P2 antagonists, suramin and PPADS, on the extent of oxygen-induced retinal neovascularization was analyzed. RESULTS: In normal mice, the expression of P2Y2 receptors was weak throughout the retina, whereas P2X2 receptor expression was detected in the outer plexiform layer. In mice treated with oxygen, P2Y2 expression was detected in the ganglion and in the nerve fiber layers, whereas P2X2 expression was found in the inner and outer plexiform layers. Oxygen-induced preretinal neovascularization was strongly inhibited by the P2 antagonists, suramin (p<0.05) and PPADS (p<0.05), and this was accompanied by a down-regulation of P2X2 receptor expression in the inner plexiform layer in suramin-treated mice. CONCLUSIONS: The data suggest that purinergic P2 receptors are involved in neovascularization associated with OIR.     

Involvement of fibroblast growth factors in choroidal angiogenesis and retinal vascularization

Fibroblast growth factors such as FGF-2 are potent mitogens for endothelial cells and induce their assembly into vascular-like structures in culture and in in vivo assays. However, their putative functions during physiological vascularization are poorly documented. In this study, the eye was used as a model for analyzing the vascular defects caused by targeted FGF inhibition in transgenic mice. Choroidal and retinal vascularizations were studied by immunohistochemistry on whole-mount preparations. Soon after activation of the transgene, angiogenesis that normally occurs during the second half of gestation in the choroid was strongly inhibited resulting in poor capillary density and branching. Later retinas strikingly failed to develop a primary vascular plexus suggesting a defect in induction of vessel assembly. Hyaloid vessels that supply the retina during the fetal period did not regress at birth and later gave rise to unexpected massive neovascularization. This model illustrates major functions of FGFs at different early stages of physiological vascularization. Both the failure in hyaloid regression and the intense angiogenic invasion of endothelial cells into the retina may serve as a model for some related human ocular pathologies.     

可溶性Tie2融合蛋白玻璃体腔注射对实验性视网膜血管新生和缺血性视网膜病变的影响

目的:观察可溶性Tie2融合蛋白(soluble Tie2 fusion pro-tein,sTie-Fc)对视网膜血管新生(retinal neovascularization,RNV)的抑制作用,同时分析其对缺血性视网膜病变的影响。方法:将生后7d的C57BL/6幼鼠置于高氧箱中饲养5d取回至正常空气环境诱导RNV模型,生后12d和14d选取一眼玻璃体腔注入人sTie-Fc0.67μg,对侧眼在相同时点注入人IgG作为对照。生后17d处死动物行视网膜铺片和免疫组织化学染色检查,测量视网膜血管无灌注区的面积,计数RNV内皮细胞核数目,定量分析sTie-Fc对于缺血性视网膜病变以及实验性RNV形成的影响。结果:对照组和实验组均成功建立了RNV模型,实验组RNV的形成受到抑制(P<0.05),同时sTie-Fc也加重了RNV模型的缺血过程(P<0.05)。结论:sTie-Fc可以抑制实验性RNV的发展,提示拮抗Tie2可能成为治疗RNV有效的生物学方法之一,其可能加重缺血性视网膜病变的潜在副作用需进一步探讨。     

Involvement of NADPH oxidase and protein kinase C in endothelin-1-induced superoxide production in retinal microvessels

Redox signaling has been implicated in pathophysiological changes in the vascular system. We examined whether endothelin-1 (ET-1) increases the formation of superoxide anions in retinal microvessels. Freshly isolated retinal microvessels from rats were exposed to ET-1 (100 nM), and the intracellular superoxide formation in the retinal pericytes was assessed semi-quantitatively by time-lapse fluorometric analyses using hydroethidine. The receptor mechanisms were determined by BQ-123 and BQ-788, receptor antagonists for ET_A and ET_B receptors, respectively, and also by IRL-1620, a selective agonist for ET_B receptors. In addition, the changes induced by adding apocynin (10 μM), myr-PKC (1.0 μM), allopurinol (100 μM), rotenone (10 μM), or L-NAME (100 μM) with ET-1 were evaluated. Microvessels were incubated with phorbol 12-myristate 13-acetate (PMA, 10 nM), a protein kinase C (PKC) activator. Fluorometric analyses showed ethidium fluorescence-positive regions that coincided well with the location of retinal pericytes. The intracellular superoxide levels were significantly increased after addition of ET-1 (100 nM), and this elevation was suppressed by apocynin or myr-PKC. Other enzyme inhibitors including L-NAME had no effect. The ET-1-induced increase of superoxide was significantly suppressed by BQ-123 (1.0 μM), while effects of adding BQ-788 (1.0 μM) were insignificant. IRL-1620 (100 nM) did not increase superoxide formation significantly. PMA (10 nM) mimicked the effect of ET-1. These results suggest that ET-1 increases the formation of superoxides in the retinal microvascular pericytes most likely by activating NADPH oxidase through ET_A receptors. The activation of PKC may be involved in the mechanism. Thus, ET-1 may augment its vasoconstrictive effects through the formation of superoxide, which may impair the bioavailability of nitric oxide in the retinal microvasculature.     

MMP-2和VEGF在视网膜新生血管的表达

目的检测基质金属蛋白酶-2(matrix metalloproteinases-2,MMP-2)和血管内皮生长因子(vascular endothelial growthfactor,VEGF)在视网膜新生血管中的表达,探讨两者的相互关系。方法 取7 d龄C57BL/6J小鼠40只,随机分为高氧组和对照组,每组各20只。建立高氧诱导的视网膜新生血管小鼠模型。采用ADP酶组织化学染色、HE染色和免疫组化方法分别观察2组视网膜血管的变化、计数突破视网膜内界膜的内皮细胞核数目和检测MMP-2、VEGF的表达。结果 高氧组视网膜铺片可见大量视网膜新生血管形成,对照组未见新生血管形成;高氧组突破视网膜内界膜内皮细胞核数目为14(14.230±5.388),与对照组数目0(0.110±0.386)相比差异有统计学意义(t=23.537,P<0.001);对照组和高氧组视网膜组织中VEGF的积分光密度值分别为36.81±14.60、60.85±24.55,差异有统计学意义(t=3.348,P<0.01);对照组和高氧组视网膜组织中MMP-2的积分光密度值分别为16.33±4.13、21.12±6.29,差异有统计学意义(t=3.160,P<0.01)。高氧组视网膜组织中MMP-2和VEGF的表达呈正相关(r=0.633,P<0.01)。结论 在ROP小鼠模型的视网膜组织中,VEGF、MMP-2的表达同步升高,二者的高表达可能与视网膜新生血管形成密切相关。     

整合连接激酶在血管内皮生长因子诱导视网膜新生血管形成的信号传导通路中的作用

目的 观察整合连接激酶（ILK）在血管内皮生长因子（VEGF）诱导的视网膜新生血管形成过程中的作用。方法 在体外培养的视网膜脉络膜血管内皮细胞（RF/6A细胞系）中，用LY294002抑制ILK活性或siRNA转染敲减ILK表达后，检测ILK对VEGF诱导的细胞黏附、增生、迁移及内皮细胞管状结构形成的作用；并在动物模型水平观察LY294002抑制ILK活性后对视网膜新生血管形成的影响。结果 正常对照组、VEGF处理组、LY294002抑制组、siRNA转染组细胞黏附实验结果分别为（0.0726±0.01961）、（0.1137±0.02631）、（0.0837±0.01503）、（0.0853±0.02454），VEGF处理组与正常对照组比较，差异有统计学意义（t =4.211，P<0.01）；LY294002抑制组以及siRNA转染组与VEGF处理组相比，差异有统计学意义(t =3.074 和2.91，P<0.01）。正常对照组、VEGF处理组、LY294002抑制组细胞增生实验结果分别为（0.4162±0.1392）、（0.6412±0.2420）、（0.4476±0.1834），VEGF处理组较正常对照组比较，差异有统计学意义（t=2.608，P<0.05）；LY294002抑制组与VEGF处理组相比，差异也有统计学意义（t=2.244，P<0.05）。正常对照组、VEGF处理组、LY294002抑制组细胞迁移实验结果分别为（83.66±30.283）、（248±74.748）、（138.5±38.167），VEGF处理组与正常对照组比较，差异有统计学意义（t=5.436，P<0.01）；LY294002抑制组与VEGF处理组相比，差异也具有统计学意义（t=3.682，P<0.01）。血管内皮细胞管状结构形成实验显示，ILK活性或表达受到抑制后无明显管状结构形成。动物实验显示腹膜下注射LY294002使视网膜后极部无灌注区面积从（62798±16995.62）μm2增加至（84722.65±10435.01） μm2，二者比较，差异有统计学意义（t=3.476，P<0.01）。 结论 应用LY294002抑制ILK活性或siRNA转染敲减ILK表达使细胞黏附率、细胞增生率及细胞迁移率均显著下降。ILK通过参与调控VEGF诱导的视网膜血管内皮细胞黏附、增生、迁移及内皮细胞管状结构形成过程而对VEGF诱导的视网膜新生血管形成过程发挥着重要作用。     

Focal adhesion kinase overexpression induces enhanced pathological retinal angiogenesis

PURPOSE: Focal adhesion kinase (FAK) is involved in processes integral to angiogenesis, such as cell growth, survival, and migration. FAK is activated by angiogenic growth factors, such as insulin-like growth factor (IGF)-I, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF). The study was conducted to determine whether overexpression of FAK or FAK-related nonkinase (FRNK), an inhibitor of FAK, could influence human retinal endothelial cell (HREC) migration and in vivo angiogenesis. METHODS: Migration in response to a combination of growth factors was examined in transfected HRECs overexpressing FAK or FRNK. The effect of FAK or FRNK overexpression on preretinal neovascularization was examined in a mouse model of oxygen-induced retinopathy. RESULTS: Overexpression of FAK in HRECs resulted in a 102% +/- 13% increase (P = 1.4 x 10(-4)) in cell migration, whereas overexpression of FRNK resulted in a 20% +/- 8% decrease (P = 0.01). Overexpression of FAK in mouse eyes led to formation of numerous large vascular tufts resembling glomeruli and a 57% +/- 7% increase in preretinal neovascularization (P = 3 x 10(-9)), whereas FRNK resulted in a 55% +/- 15% reduction (P = 5 x 10(-5)). CONCLUSIONS: Modulating the FAK/FRNK system may provide a novel approach to inhibiting pathologic retinal angiogenesis.     

Specific involvement of SRC family kinase activation in the pathogenesis of retinal neovascularization

PURPOSE: Src family kinases (SFKs) are membrane-attached nonreceptor protein tyrosine kinases that link a variety of extracellular cues to intracellular signal pathways. The purpose of this study was to characterize the roles of SFKs in vascular endothelial growth factor (VEGF)-mediated retinal angiogenesis. METHODS: Primary rat retinal glial Müller cells and bovine and human retinal microvascular endothelial cells (RMECs) were used in the in vitro studies. A rat model of retinopathy of prematurity (ROP) was used in the in vivo studies. RESULTS: In vitro, SFKs were essential for hypoxia-induced VEGF expression in Müller cells and for VEGF signaling in RMECs. However, neither process required significant further phosphorylation of the SFK activation loop Tyr416. In vivo, in a rat model of ROP, a pronounced increase of retinal SFK Tyr416 phosphorylation was observed that was specifically associated with pathologic angiogenesis. These retinas also expressed significantly higher levels of VEGF than did those in healthy controls. Immunohistochemical analysis indicated that Müller cells were the major source of the elevated level of phospho-SFK Tyr416. Intravitreous injection of a selective SFK inhibitor, PP2, significantly reduced retinal VEGF and retinopathy in the ROP model, indicating that SFKs acted as important regulators in abnormal retinal angiogenesis. CONCLUSIONS: Together, these data suggest that SFK activation through a Tyr416-dependent mechanism may be an important factor in the pathogenesis of retinal neovascularization.     

MMP-2和VEGF在视网膜新生血管中的表达及意义

目的探讨基质金属蛋白酶-2（MMP-2）和血管内皮生长因子（VEGF）在视网膜新生血管中的表达及意义。方法取C57BL/6J小鼠60只,随机分为高氧组和正常组,各30只。以高浓度氧诱导小鼠建立视网膜新生血管模型。采用ADP酶视网膜铺片、苏木精-伊红染色及免疫组织化学法分别观察视网膜血管的改变、计数视网膜新生血管内皮细胞数并检测MMP-2、VEGF蛋白的表达。结果高氧组视网膜可见大量新生血管形成;突破视网膜内界膜的新生血管内皮细胞核数为（33.51±2.55）个,与对照组相比差异有统计学意义（t=9.345,P〈0.05）。高氧组与对照组比较,MMP-2、VEGF蛋白在神经节细胞层、内丛状层、内核层和突破视网膜内界膜的新生血管中高表达,且二者表达呈正相关（r=0.825,P〈0.05）。结论MMP-2、VEGF共同促进视网膜新生血管的形成,且二者可能具有协同作用。     

Involvement of protein kinase C in phagocytosis of human retinal pigment epithelial cells and induction of matrix metalloproteinase secretion

Protein kinase C (PKC) is involved in cell activation. We investigated PKC-mediated pathways and secretion of matrix metalloproteinases (MMPs) in phagocytosis by human retinal pigment epithelial cells (RPE). We used time-resolved fluorometry for europium-labeled microsphere uptake and gel zymography to assay the influence of PKC modulators. PKC inhibitors blocked phagocytosis by RPE. ARPE-19, a human RPE-cell line, showed reduced secretion of MMP-2, although MMP-9 secretion by PKC activation was conserved in both cell types, namely in the primary RPEs and in the RPE-cell line. Particle uptake by RPE cells requires activation of PKC; the use of PKC inhibitors as new anticancer drugs may possibly cause ocular side-effects.     

Platelet-induced retinal neovascularization in leukemia

Retinal changes resembling those of background diabetic or hypertensive retinopathy commonly occur in leukemia, whereas retinal neovascularization is rare. When neovascularization does occur, it is usually the result of hyperviscosity caused by a greatly increased number of circulating leukocytes. A 42-year-old woman with diabetes mellitus developed chronic myelocytic leukemia and peripheral retinal neovascularization. The hyperviscosity leading to the neovascularization was probably caused by an increased number of circulating platelets.     

Retinitis pigmentosa and retinal neovascularization

Four patients with retinitis pigmentosa and either disc or peripheral retinal neovascularization with recurrent vitreous hemorrhage are described. One patient with peripheral retinal neovascularization also had rubeosis and neovascular glaucoma. The effects of relative hyperoxia on the retinal microcirculation in retinitis pigmentosa as well as intraocular inflammation may account for such changes. Laser photocoagulation appears effective in preventing vitreous hemorrhage in these patients, but systemic administration of corticosteroids did not cause the new vessels to regress.     

Reduced retinal angiogenesis in MMP-2-deficient mice

PURPOSE: To study the putative role of endogenous matrix metalloproteinases (MMPs) in retinal neovascularization, an established mouse model was used to compare the retinal neovascularization observed in wild-type mice with that in mice without the MMP-2 or -9 genes. METHODS: C57Bl/6 (MMP-2(+/+) and -9(+/+)), MMP-2-deficient (MMP-2(-/-)), and MMP-9-deficient (MMP-9(-/-)) mice were used. After oxygen-induced retinopathy was induced in the mice, their eyes were rapidly removed and frozen in optimal cutting temperature embedding compound. Sections were histochemically stained with specific markers for vascular cells and angiogenesis-related factors. The area of new retinal vessels was measured using image-analysis software and compared between groups. RESULTS: Retinal neovascularization was not significantly different between wild-type and MMP-9(-/-) mice. The MMP-2(-/-) mice had significantly less extraretinal neovascularization than did wild-type mice. The mean number of extraretinal neovascular buds per cross section was significantly lower in MMP-2(-/-) mice than in wild-type mice (P < 0.05). The expression of other angiogenesis-related factors, vascular endothelial growth factor and pigment epithelium-derived factor, was not different between wild-type and MMP-2(-/-) mice. CONCLUSIONS: MMP-2 may be essential in the regulation of retinal neovascularization. Pharmacologic intervention using MMP inhibitors may be a future therapeutic approach for angiogenic retinal diseases.     

酪氨酸激酶及其抑制剂在视网膜新生血管形成机制及治疗中的研究进展

视网膜新生血管的发生与发展涉及多种生长因子及其受体复杂的生物学过程,许多生长因子的受体均为蛋白酪氨酸激酶受体.蛋白酪氨酸激酶在新生血管的发生与发展中起非常重要的作用,生长因子生物学活性的表达需要与其靶细胞受体结合并激活受体酪氨酸激酶,引起其结构改变从而产生酶催化效应,引起生物学效应,促进新生血管形成.通过寻找选择性的酪氨酸酶抑制剂,来阻断或调控由于这些信号通路异常激活产生的疾病,是抗新生血管药物的研究策略.     

MMP-2和PEDF在氧诱导小鼠视网膜新生血管中的表达

目的探讨基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)和色素上皮衍生因子(pigment epithelium derived factor,PEDF)在氧诱导小鼠视网膜新生血管中的表达和意义。方法随机选取80只健康C57BL/6J小鼠进行分组,取对照组(40只)和高氧组(40只)小鼠眼球作ADP酶视网膜铺片、病理切片及免疫组织化学法检测,观察视网膜血管的改变,计算视网膜新生血管突破内界膜的内皮细胞核数及检测MMP-2、PEDF蛋白的表达。结果高氧组视网膜大量新生血管形成;新生血管突破内界膜的内皮细胞核数为(32.45±1.34)个,与对照组(1.27±0.20)个相比差异有统计学意义(t=10.345,P<0.05)。高氧组与对照组相比,MMP-2蛋白在神经节细胞层、内丛状层、内核层和突破视网膜内界膜的新生血管中高表达(P<0.05);PEDF蛋白在神经纤维层、神经节细胞层、内丛状层、感光细胞层及视网膜色素上皮细胞层低表达(P<0.05)。两者表达呈显著负相关(r=-0.785,P<0.05)。结论 MMP-2和PEDF共同维持视网膜新生血管的形成。     

Inhibition of retinal neovascularization by soluble EphA2 receptor

Eph receptor tyrosine kinases (RTKs) and their ligands, known as ephrins, play an important role in vascular remodeling during embryogenesis, but their functions in adult angiogenesis are just beginning to be investigated. In this report, we investigated the effect of blocking EphA receptor activation on VEGF-induced angiogenic responses of cultured retinal endothelial cells and on retinal neovascularization in a rodent model of retinopathy of prematurity (ROP). Soluble EphA2-Fc receptors inhibited ephrin-A1 ligand or VEGF-induced BRMEC migration and tube formation without affecting proliferation in vitro. Since EphA2-Fc receptors can inhibit activation of multiple EphA receptors, the specific role of EphA2 receptor in angiogenesis was further investigated in EphA2-deficient endothelial cells. Loss of EphA2 in endothelial cells leads to defective cell migration and assembly in response to either ephrin-A1 or VEGF. Finally, a significant reduction in the severity of abnormal retinal neovascularization was observed in the eyes treated with soluble EphA2-Fc receptors, yet the normal total retinal vascular area was not significantly changed. Because soluble Eph receptor significantly inhibited pathologic retinal angiogenesis without affecting normal intraretinal vessels, it may be a promising agent for treatment of retinal angiogenesis in a number of human ocular diseases.     

Pathogenesis of retinal neovascularization]

Presented are the causes and actual opinions on the pathogenesis of retinal neovascularization. The ischaemic hypothesis of neovascularization is particularly discussed; it is commonly accepted to day as the most probable hypothesis.     

内皮祖细胞与视网膜新生血管

视网膜新生血管可见于多种眼部疾病,是引起视力损害的重要原因之一.新近研究发现,循环中的血管内皮祖细胞在视网膜新生血管的形成中起重要作用,并影响新生血管的严重程度.这一发现为视网膜新生血管的防治提供了新思路.针对血管内皮祖细胞的动员、趋化和黏附等治疗视网膜新生血管的方法正在探索中.