Wong Wai T, Rex Tonia S, Auricchio Alberto, Maguire Albert M, Chung Daniel, Tang Waixing, Bennett Jean
F. M. Kirby Center for Molecular Ophthalmology, Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia 19104-6069, USA.
Mol Vis. 2004 Nov 8;10:837-44.
Pigment epithelium derived factor (PEDF) is a secreted protein with demonstrated anti-angiogenic properties, and with potential application for the treatment of neovascular disease. Delivery of pigment epithelium derived factor to the retina via virus mediated gene transfer has been shown to inhibit neovascularization in a number of experimental models. While pigment epithelium derived factor is endogenously expressed in the retina, its role in guiding normal vessel development and growth is not yet known. This study aimed to determine whether over-expression of pigment epithelium derived factor alters the normal pattern of retinal vessel development.
Neonatal (age postnatal day 2 (P2)) CD1 mice were injected subretinally unilaterally with AAV2/1.CMV.PEDF while contralateral eyes were injected subretinally with AAV2/1.CMV.EGFP as control. Cohorts of animals were sacrificed at P7 to P21 and the retinal vasculature was co-labeled through fluorescein-dextran perfusion and immunohistochemistry. Vascular size, localization, and structure were analyzed using light and confocal microscopy. Additional cohorts were use to obtain quantitative levels of pigment epithelium derived factor protein through ELISA.
The extent of vessel growth from the optic disk to periphery over time (i.e., the radius of retinal vasculature), and the area of expansion of the neural retina were unaffected by over-expression of pigment epithelium derived factor to levels at least 3.5 fold higher than endogenous levels. The thicknesses of the various retinal layers were similar in AAV2/1.CMV.PEDF treated and control injected eyes. Three dimensional analysis of confocal images shows a slight delay in the rate of growth of vasculature into the deeper layers of the retina in pigment epithelium derived factor treated eyes compared to EGFP treated control eyes. However, the normal differentiation of vessels into arterioles, and venules, and the formation of a capillary network continued to occur, achieving normal and complete maturation of vascular structure by P21.
Over-expression of pigment epithelium derived factor in the developing retina exerted no marked or permanent effects on retinal vessel growth and differentiation. The findings are relevant to the safety of the potential therapeutic use of pigment epithelium derived factor in human retinal disease.
色素上皮衍生因子（PEDF）是一种具有抗血管生成特性的分泌蛋白，在治疗新生血管疾病方面具有潜在应用价值。通过病毒介导的基因转移将色素上皮衍生因子递送至视网膜，已在多个实验模型中显示出可抑制新生血管形成。虽然色素上皮衍生因子在视网膜中内源性表达，但其在引导正常血管发育和生长中的作用尚不清楚。本研究旨在确定色素上皮衍生因子的过表达是否会改变视网膜血管发育的正常模式。
对出生后第2天（P2）的新生CD1小鼠单侧视网膜下注射AAV2/1.CMV.PEDF，对侧眼视网膜下注射AAV2/1.CMV.EGFP作为对照。在P7至P21处死动物群体，通过荧光素-葡聚糖灌注和免疫组织化学对视网膜血管系统进行共标记。使用光学显微镜和共聚焦显微镜分析血管大小、定位和结构。使用额外的动物群体通过酶联免疫吸附测定（ELISA）获得色素上皮衍生因子蛋白的定量水平。
随着时间推移，从视盘到周边的血管生长范围（即视网膜血管系统的半径）以及神经视网膜的扩展面积，不受色素上皮衍生因子过表达的影响，其过表达水平至少比内源性水平高3.5倍。在接受AAV2/1.CMV.PEDF治疗的眼睛和注射对照的眼睛中，各视网膜层的厚度相似。共聚焦图像的三维分析显示，与接受EGFP治疗的对照眼睛相比，接受色素上皮衍生因子治疗的眼睛中，血管向视网膜深层生长的速率略有延迟。然而，血管正常分化为小动脉和小静脉以及毛细血管网络的形成仍继续发生，到P21时血管结构实现正常且完全成熟。
发育中的视网膜中色素上皮衍生因子的过表达对视网膜血管生长和分化没有明显或永久性影响。这些发现与色素上皮衍生因子在人类视网膜疾病潜在治疗应用中的安全性相关。
