色素上皮衍生因子可抑制缺血诱导的视网膜新生血管形成以及血管内皮生长因子诱导的迁移和生长。

Pigment epithelium-derived factor suppresses ischemia-induced retinal neovascularization and VEGF-induced migration and growth.

作者信息

Duh Elia J, Yang Hoseong S, Suzuma Izumi, Miyagi Masaru, Youngman Elaine, Mori Keisuke, Katai Miyuki, Yan Lin, Suzuma Kiyoshi, West Karen, Davarya Shekar, Tong Patrick, Gehlbach Peter, Pearlman Joel, Crabb John W, Aiello Lloyd P, Campochiaro Peter A, Zack Donald J

机构信息

Department of Ophthalmology, Johns Hopkins University School of Medicine, Maumenee 809, 600 N. Wolfe Street, Baltimore, MD 21287, USA.

出版信息

Invest Ophthalmol Vis Sci. 2002 Mar;43(3):821-9.

PMID:11867604

Abstract

PURPOSE

To determine the effect of pigment epithelium-derived factor (PEDF) in a mouse model of ischemia-induced retinal neovascularization and on vascular endothelial growth factor (VEGF)--induced migration and growth of cultured microvascular endothelial cells.

METHODS

Human recombinant PEDF was expressed in the human embryonic kidney 293 cell line and purified by ammonium sulfate precipitation and cation exchange chromatography. C57BL/6 mice were exposed to 75% oxygen from postnatal day (P)7 to P12 and then returned to room air. Mice received intravitreal injections of 2 microg PEDF in one eye and vehicle in the contralateral eye on P12 and P14. At P17, mice were killed and eyes enucleated for quantitation of retinal neovascularization. The mitogenic and motogeneic effects of VEGF on cultured bovine retinal and adrenal capillary endothelial cells were examined in the presence or absence of PEDF, using cell counts and migration assays.

RESULTS

Two species of human recombinant PEDF, denoted A and B, were purified to apparent homogeneity. PEDF B appeared to comigrate on SDS-PAGE with PEDF from human vitreous samples. Changes in electrophoretic mobility after peptide-N-glycosidase F (PNGase F) digestion suggest that both PEDF forms contain N-linked carbohydrate. Analyses of the intact proteins by liquid chromatography-electrospray mass spectrometry (LC-ESMS) revealed the major molecular weight species for PEDF A (47,705 +/- 4) and B (46,757 +/- 5). LC-ESMS analysis of tryptic peptides indicated that PEDF A and B exhibit differences in glycopeptides containing N-acetylneuraminic acid (NeuAc) and N-acetylhexosamine (HexNAc). Intravitreal administration of either species of PEDF significantly inhibited retinal neovascularization (83% for PEDF A and 55% for PEDF B; P = 0.024 and 0.0026, respectively). PEDF A and B (20 nM) suppressed VEGF-induced retinal microvascular endothelial cell proliferation by 48.8% and 41.4%, respectively, after 5 days (P < 0.001) and VEGF-induced migration by 86.5% +/- 16.7% and 78.1% +/- 22.3%, respectively, after 4 hours (P = 0.004 and P = 0.008, respectively).

CONCLUSIONS

These data indicate that elevated concentrations of PEDF inhibit VEGF-induced retinal endothelial cell growth and migration and retinal neovascularization. These findings suggest that localized administration of PEDF may be an effective approach for the treatment of ischemia-induced retinal neovascular disorders.

摘要

目的

确定色素上皮衍生因子（PEDF）在缺血诱导的视网膜新生血管形成小鼠模型中以及对血管内皮生长因子（VEGF）诱导的培养微血管内皮细胞迁移和生长的影响。

方法

人重组PEDF在人胚肾293细胞系中表达，通过硫酸铵沉淀和阳离子交换色谱法纯化。C57BL/6小鼠从出生后第7天（P7）至P12暴露于75%氧气中，然后放回室内空气中。在P12和P14时，小鼠一只眼玻璃体内注射2μg PEDF，对侧眼注射赋形剂。在P17时，处死小鼠并摘除眼球以定量视网膜新生血管形成。使用细胞计数和迁移试验，在有或无PEDF存在的情况下，检测VEGF对培养的牛视网膜和肾上腺毛细血管内皮细胞的促有丝分裂和促运动作用。

结果

两种人重组PEDF，分别记为A和B，纯化至表观均一。PEDF B在SDS-PAGE上似乎与人玻璃体样品中的PEDF迁移一致。肽-N-糖苷酶F（PNGase F）消化后电泳迁移率的变化表明两种PEDF形式均含有N-连接碳水化合物。通过液相色谱-电喷雾质谱（LC-ESMS）对完整蛋白的分析揭示了PEDF A（47,705±4）和B（46,757±5）的主要分子量种类。胰蛋白酶肽段的LC-ESMS分析表明，PEDF A和B在含有N-乙酰神经氨酸（NeuAc）和N-乙酰己糖胺（HexNAc）的糖肽方面存在差异。玻璃体内注射任何一种PEDF均显著抑制视网膜新生血管形成（PEDF A为83%，PEDF B为55%；P分别为0.024和0.0026）。5天后，PEDF A和B（20 nM）分别将VEGF诱导的视网膜微血管内皮细胞增殖抑制48.8%和41.4%（P<0.001），4小时后分别将VEGF诱导的迁移抑制86.5%±16.7%和78.1%±22.3%（P分别为0.004和0.008）。