Effect of over-expression of pigment epithelium derived factor (PEDF) on developing retinal vasculature in the mouse.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Wong
Wai T
WT
F. M. Kirby Center for Molecular Ophthalmology, Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia 19104-6069, USA.
Rex
Tonia S
TS
Auricchio
Alberto
A
Maguire
Albert M
AM
Chung
Daniel
D
Tang
Waixing
W
Bennett
Jean
J
D000818:Animals; D000831:Animals, Newborn; D002454:Cell Differentiation; D000229:Dependovirus / Q000235:genetics; D004730:Endothelium, Vascular / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005136:Eye Proteins / Q000502:physiology*; D015870:Gene Expression; D005822:Genetic Vectors; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009414:Nerve Growth Factors / Q000502:physiology*; D009898:Optic Disk / Q000098:blood supply; D012333:RNA, Messenger / Q000378:metabolism; D012171:Retinal Vessels / Q000254:growth & development* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015843:Serpins / Q000502:physiology*; D019076:Transgenes; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D000099174:Pigment Epithelium-Derived Factor
D005136:Eye Proteins; D009414:Nerve Growth Factors; D012333:RNA, Messenger; D015843:Serpins; D042461:Vascular Endothelial Growth Factor A; D000099174:Pigment Epithelium-Derived Factor; C467480:vascular endothelial growth factor A, mouse