The retina survival effects of PEDF require the binding to cell-surface receptors. We have identified PEDF-R, a protein encoded by the Pnpla2 gene for which PEDF is a natural ligand and is expressed in the retina and RPE cells. To better evaluate the contribution of PEDF-R to the PEDF biological activity in vivo, we generated a conditional Pnpla2 null mouse model. Heterozygous Pnpla2/LoxP (Pnpla2f/+) were intercrossed to generate homozygous Pnpla2/LoxP (Pnpla2f/f) mutant mice, carrying LoxP sites flanking a portion of the Pnpla2 gene (floxed region). These mice were then crossed with transgenic mouse lines carrying the Cre recombinase gene, in which the expression is under the control of promoters specific to the photoreceptor or RPE compartments of the eye. These crosses resulted in the generation of conditional Pnpla2-null mice specific to photoreceptors or RPE. The genomic deletion of the floxed region was confirmed by PCR genotyping. To assess the functionality of rod and cone photoreceptors after PEDF-R depletion, electroretinogram analysis was performed in conditional knockout mice of Pnpla2 specific to photoreceptors. PEDF has protective effects on light-induced photoreceptor damage. To optimize a mouse model for photoreceptor damage we exposed wild-type mice to continuous high-intensity white light followed by recovery in the dark. The effects of light treatment on retina morphology were evaluated by optical coherence tomography. PEDF also has a protective effect on photoreceptors in mouse models of inherited retina degeneration. C57BL/6Jrd10/rd10 mouse strain, (abbreviated rd10) is an established mouse model that carries a missense point mutation in the rod cGMP phosphodiesterase gene (Pde6b) resulting in genetically induced photoreceptor degeneration. This mutation abolishes the PDE6 enzymatic activity in photoreceptors. Retina explant cultures from rd10 mice were developed and optimized for testing the retinoprotective activity of PEDF and peptides, as well as in the presence of PEDF/PEDF-R blocking peptide. TUNEL assays were performed on fixed tissues of PEDF-treated retina explants. Antiapoptotic effects were observed in PEDF-treated retina explants from rd10 mice. Specific phosphodiesterase 6 inhibitors in wild type retina explants can also replicate the degeneration typically ascribed to rd10 mice. Antiapoptotic effects were also observed in PEDF-treated retinas exposed to phosphodiesterase inhibitors. PEDF-R protein and Pnpla2 expression levels were determined in retinas from rd10 animals at different time points between 15-24 days old by western blotting of tissue extracts and RT-PCR of laser-captioned retina sections, respectively. Primary RPE cell cultures were prepared from eyes of rd10 and wild type mice at different ages to determine the levels of PEDF protein secreted by the cultured cells. To evaluate the role of PEDF-R in mediating PEDF pro-survival activity in genetically compromised photoreceptors, we crossed the photoreceptor-specific Pnpla2-KO mice with rd10 mice. This cross resulted in mouse models carrying photoreceptors without PEDF-R and with the rd10 mutation. PEDF neuroprotective and antiangiogenic functions are restricted to 2 regions of the protein. A 44-mer peptide is associated with neuroprotection, whereas the antiangiogenic activity is mediated by a 34-mer. With the ultimate goal of developing and characterizing novel PEDF-related therapeutic agents, we constructed expression plasmids containing the full length cDNA of SERPINF1 gene with mutations to alter two single amino acids within the 44-mer region of PEDF. Peptides containing these 2 alterations were previously assessed in vitro and found to affect PEDF binding affinity for PEDF-R. BHK cells were transfected with these plasmids to overexpress the mutated SERPINF1 and obtain recombinant proteins. Purification protocols were developed for the recombinant altered PEDF prooteins from the conditioned media of stably transfected BHK cells with the PEDF expression vectors. Purified recombinant proteins were produced to evaluate their biological function in vivo.
| Polato, Federica; Becerra, S Patricia (2016) Pigment Epithelium-Derived Factor, a Protective Factor for Photoreceptors in Vivo. Adv Exp Med Biol 854:699-706 |
| Kenealey, Jason; Subramanian, Preeti; Comitato, Antonella et al. (2015) Small Retinoprotective Peptides Reveal a Receptor Binding Region on Pigment Epithelium-derived Factor. J Biol Chem : |
| Amaral, Juan; Becerra, S Patricia (2010) Effects of human recombinant PEDF protein and PEDF-derived peptide 34-mer on choroidal neovascularization. Invest Ophthalmol Vis Sci 51:1318-26 |
| Samtani, Sonia; Amaral, Juan; Campos, Maria M et al. (2009) Doxycycline-mediated inhibition of choroidal neovascularization. Invest Ophthalmol Vis Sci 50:5098-106 |
