Ge Hong-Yan, Xiao Nan, Yin Xiu-Li, Fu Song-Bin, Ge Jin-Ying, Shi Yan, Liu Ping
Key Laboratory of Harbin Medical University Eye Center, Eye Hospital, First Affiliated Hospital, Harbin Medical University, Harbin, P.R. China.
Mol Vis. 2011;17:1918-28. Epub 2011 Jul 15.
Endostatin plays an important role in inhibiting corneal neovascularization (CNV). The aim of this study was to evaluate the antiangiogenic activities of lipid-mediated subconjunctival injection of the modified RGDRGD (arginine- glycin- aspartic- arginine- glycin- aspartic- endostatin gene in a rabbit model of neovascularization in vivo.
A modified human endostatin gene containing an RGDRGD motif was obtained by rapid site-directed mutagenesis. Forty New Zealand white rabbits underwent alkaline burn and developed CNV, which were randomly divided into four groups: an experimental control group, a PCI empty vector group, a PCI-endostatin group, and a PCI-RGDRGD-endostatin group. The vector, endostatin, and RGDRGD-endostatin groups received injections into the superior bulbar conjunctiva after the burn. An injection of 5 μg was given twice at 1-week intervals. Four eyes of two rabbits received neither treatment nor alkaline burn and served as absolute normal controls. The areas of CNV were monitored after 7 and 14 days. Corneas were examined by histology, and VEGF (vascular endothelial growth factor) and CD31 (platelet endothelial cell adhesion molecule-1) expression was detected by immunohistochemistry after 7 and 14 days. Retina, liver, and kidney were examined by histology, and CD38 expression in the inflammatory cells was detected by immunohistochemistry at 90 days.
Subconjunctival injection of both native endostatin and modified RGDRGD-endostatin genes resulted in a significant suppression of CNV in vivo, with modified RGDRGD-endostatin being more effective than native endostatin. The mean concentration of VEGF in the PCI-RGDRGD-endostatin group significantly decreased compared to the means in the other groups. Upon histological examination, the endostatin-treated and RGDRGD-endostatin-treated eyes showed significantly less neovascular area and fewer vessels than the control and vector-injected groups. Retinal, hepatic, and renal tissue sections were normal, and there was no inflammatory cell infiltration observed.
Native and modified endostatin can significantly inhibit CNV by suppressing the expression of VEGF. However, modified endostatin with the RGDRGD motif is far more effective than the endostatin gene in antiangiogenic activity.
内皮抑素在抑制角膜新生血管形成（CNV）中起重要作用。本研究旨在评估脂质介导的结膜下注射修饰后的RGDRGD（精氨酸 - 甘氨酸 - 天冬氨酸 - 精氨酸 - 甘氨酸 - 天冬氨酸） - 内皮抑素基因在兔体内新生血管形成模型中的抗血管生成活性。
通过快速定点诱变获得含RGDRGD基序的修饰人内皮抑素基因。40只新西兰白兔经历碱烧伤并形成CNV，随机分为四组：实验对照组、PCI空载体组、PCI - 内皮抑素组和PCI - RGDRGD - 内皮抑素组。烧伤后，载体组、内皮抑素组和RGDRGD - 内皮抑素组接受上睑结膜下注射。每隔1周注射5μg，共注射两次。两只兔子的四只眼睛既未接受治疗也未经历碱烧伤，作为绝对正常对照。在7天和14天后监测CNV面积。7天和14天后通过组织学检查角膜，并用免疫组织化学法检测血管内皮生长因子（VEGF）和血小板内皮细胞黏附分子 - 1（CD31）的表达。90天时通过组织学检查视网膜、肝脏和肾脏，并用免疫组织化学法检测炎症细胞中CD38的表达。
结膜下注射天然内皮抑素和修饰后的RGDRGD -内皮抑素基因均能在体内显著抑制CNV，修饰后的RGDRGD -内皮抑素比天然内皮抑素更有效。与其他组相比，PCI - RGDRGD -内皮抑素组中VEGF的平均浓度显著降低。组织学检查显示，与对照组和注射载体组相比，内皮抑素治疗组和RGDRGD -内皮抑素治疗组的新生血管面积显著减小，血管数量减少。视网膜、肝脏和肾脏组织切片正常，未观察到炎症细胞浸润。
天然和修饰后的内皮抑素可通过抑制VEGF表达显著抑制CNV。然而，带有RGDRGD基序的修饰内皮抑素在抗血管生成活性方面远比内皮抑素基因有效。
