Effect of Over-expression of Pigment Epithelium-Derived Factor (PEDF) on Developing Retinal Vasculature in the Mouse Eye

Abstract

Abstract: : Purpose: PEDF is a secreted protein with demonstrated anti-angiogenic properties. PEDF is endogenously expressed in the developing retina and is temporally regulated across the period of vessel development. Delivery of PEDF to the retina via virus-mediated gene transfer has been shown to inhibit neovascularization in a number of experimental models. It is therefore of interest to discover if over-expressing PEDF by similar means during development results in an arrest in normal vasculature development. Methods: Adeno-associated viral vector AAV2/1 containing cDNA encoding PEDF or enhanced green fluorescent protein (EGFP) driven by a CMV promoter were produced. Neonatal CD1 mice aged from P0 to P4 were given a subretinal injection of the vector containing a PEDF construct (0.5 to 1 µl of 6 x 10¹² genome copies/µl) in the right eye, and the EGFP control vector in the left eye. The animals were sacrificed at P12 to P14 and fluorescein dextran flat-mounts of treated and control eyes were prepared and compared. Some flat-mounts were also co-labeled with fluorescent-conjugates of Griffonia simplicifolia isolectin GS-IB4 that specifically labels endothelial cells of the developing blood vessels. Results: Transgene expression was observed in flat-mounts analyzed. Over-expression of AAV2/1 vector-driven PEDF had no significant effect on retinal vasculature development when compared to the control vector in the contralateral eye. There were no marked differences in extent of vessel growth from the optic disk to the periphery. Vessel growth into the deeper layers of the retina continued to occur, and the differentiation of vessels into arterioles and venules and the formation of a capillary network occurred to similar extents compared to controls. Conclusions: Over-expression of PEDF in the developing retina by AAV2/1-driven gene expression exerted no marked effect on various aspects of retinal vessel growth and differentiation. The developmental role of PEDF may be more related to limiting vessel growth to the confines of the developing retina and less with the shaping the architecture of vascular trees or the regulation of vessel differentiation.

Keywords: retinal development • gene transfer/gene therapy • retinal neovascularization