Abstract
Purpose: :
The purpose of this study was to investigate whether lens epithelium derived growth factor (LEDGF/p75), a survival factor for photoreceptor and retinal pigment epithelial cells, and its splice variant (p52) could be delivered to the retina by the transscleral pathway.
Methods: :
Transscleral transport of His–LEDGF/p75 was analyzed in vitro using isolated bovine sclera in side by side diffusion chambers. A 10 µg/ml solution was used for transport studies and the cumulative % transported was evaluated at the end of 4 hrs. A direct infusion mass spectrometric analysis was used for the detection and quantification of histidine LEDGF (His–LEDGF/His–p75) and histidine and green fluorescent protein tagged p52 (His–GFP–p52). For in vivo studies, a 10 µl solution of His–GFP–p52 (1.1 mg/ml) was injected into the posterior subconjunctival space of one eye of anesthetized male Sprague Dawley rats. The rats were sacrificed at 4 hrs after subconjunctival administration. The eyes were enucleated, embedded in optimal cutting temperature medium, and frozen. Seven µm sections were cut and visualized by confocal microscopy. Cellular entry of His–GFP–p52 and its expression pattern from a plasmid construct was also determined using 661w (retinal photoreceptors of murine origin) cells and confocal microscopy.
Results: :
The standard curves for His–GFP–p52 and His–LEDGF were linear in the ranges 5–125 ng and 5–60 ng, respectively. His–LEDGF was transported across the sclera, with the cumulative % transport being 3.2 ± 0.53 % at 4 hrs. Following subconjunctival administration, His–GFP–p52 was visualized in the retina. There was an intense fluorescence in the retinal pigment epithelium, outer nuclear layer, inner nuclear layer, and the ganglion cell layer. Low intensity fluorescence was detected in the retina of the contralateral eye. Exogenous His–GFP–p52 was localized around the nucleus in 661w cells within 8 hrs. His–GFP–p52 expressed using a plasmid construct was localized in the nucleus at 24 hrs after transfection.
Conclusions: :
LEDGF permeates across isolated bovine sclera. p52 reaches the retina following subconjunctival administration to rats. This delivery is most likely through the transscleral pathway. Further, p52 enters retinal cells in vitro.
Keywords: retinal degenerations: cell biology • proteomics • protein structure/function