Antiangiogenesis Effects of Endostatin in Retinal Neovascularization
Antiangiogenesis Effects of Endostatin in Retinal Neovascularization
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DOI：
10.1089/jop.2012.0225
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发表时间：
2013-09-01
影响因子：
2.3
通讯作者：
Li, Xiao-xin
中科院分区：
文献类型：
--
作者：
Bai, Yu-jing;Huang, Lv-zhen;Li, Xiao-xin
目的：病理性视网膜血管生成是视力丧失的主要原因。内皮抑素是一种天然的抗血管生成的抗肿瘤蛋白，广泛应用于肿瘤研究。本研究旨在探讨内皮抑素（endostatin）对视网膜新生血管的预防作用及其机制。方法：采用人脐静脉内皮细胞（HUVECs）进行体外实验。将HUVEC与内皮抑素或血管内皮生长因子（VEGF）和内皮抑素孵育不同的时间点。分别使用Cell Counting Kit-8测定、Transwell测定、流式细胞术和Matrigel测定进行细胞增殖、迁移、细胞周期和管形成研究。酶联免疫吸附试验（ELISA）检测不同时间点HUVECs分泌VEGF和色素上皮衍生因子（PEDF）蛋白的情况。鼠氧诱导视网膜病变（OIR）模型用于体内研究。将7日龄C57 BL/6 J幼仔（p7）暴露于75%氧气中5天。在p12，将动物返回到正常大气中，并立即玻璃体内注射1.5 μ L的5 mg/mL内皮抑制素溶液。在p18时，用荧光素-葡聚糖-FITC灌注小鼠，并将其视网膜平放以测量非灌注面积。结果：内皮抑素在体外呈剂量依赖性抑制HUVEC增殖，在含VEGF的培养液中也能抑制HUVEC增殖。此外，内皮抑制素可以抑制HUVEC中的迁移、管形成和VEGF分泌，同时还在几个时间点诱导HUVEC中的凋亡。与对照组相比，这些效果具有统计学意义（P < 0.05）。在体内，单次玻璃体内注射内皮抑素使视网膜非灌注面积从对照组的30%减少到治疗组的23%（P < 0.0001）。结论：内皮抑素在体外和体内均具有明显的抑制血管生成的作用。抑制作用可能至少部分是由于PEDF/VEGF比率的恢复。这些数据表明，内皮抑素可以提供一个创新的药物策略，用于预防视网膜新生血管。
Purpose: Pathological retinal angiogenesis is a major cause of vision loss. Endostatin is a natural antiangiogenesis antitumor protein that is widely used in cancer studies. In this study, we investigated the efficacy and potential mechanisms of endostatin for the prevention of retinal neovascularization both in vitro and in vivo.Methods: Human umbilical vein endothelial cells (HUVECs) were used for the in vitro studies. HUVECs were incubated with endostatin or the vascular endothelial growth factor (VEGF) and endostatin for different time points. Cell proliferation, migration, cell cycling, and tube formation studies were carried out using a Cell Counting Kit-8 assay, a Transwell assay, flow cytometry, and a Matrigel assay, respectively. Enzyme-Linked Immunosorbent Assay (ELISA) was used to study VEGF and pigment epithelial-derived factor (PEDF) protein secretion from the HUVECs at different time points. A murine oxygen-induced retinopathy (OIR) model was used for the in vivo studies. Seven-day-old C57BL/6J pups (p7) were exposed to 75% oxygen for 5 days. On p12, the animals were returned to a normal atmosphere and were immediately injected intravitreously with 1.5 mu L of a 5 mg/mL endostatin solution. At p18, the mice were perfused with fluorescein-dextran-FITC, and their retinas were flat mounted to measure the nonperfused area. Retinal VEGF and PEDF levels were also measured by ELISA Kits in the OIR mice at p18.Results: In vitro, endostatin inhibited HUVEC proliferation in a dose-dependent manner and also inhibited HUVEC proliferation in a VEGF-containing medium. Additionally, endostatin can inhibit migration, tube formation, and VEGF secretion in HUVECs, while also inducing apoptosis in HUVECs at several time points. These effects were statistically significant when compared to the control group (P < 0.05). In vivo, a single intravitreous injection of endostatin reduced the retinal nonperfused area from 30% in the control group to 23% in the treatment group (P < 0.0001). Intravitreous injection of endostatin reduced VEGF levels in retinas, while it increased PEDF levels.Conclusions: Endostatin showed convincing inhibitory effects on angiogenesis both in vitro and in vivo. The inhibitory effects may be, at least partly, resulted from the restoration of the PEDF/VEGF ratio. These data suggest that endostatin could offer an innovative pharmaceutical strategy for the prevention of retinal neovascularization.