We completed the studies on the effects of doxycycline (a bacteriostatic antibiotic with anti-metalloproteolytic and anti-angiogenic properties) on choroidal neovascularization (CNV). Doxycycline was administered orally to rats, and plasma levels of PEDF, matrix metalloproteinases MMP-2 and MMP-9 proteins were measured by ELISA. Experimental CNV was induced in rats. Volumes of laser-induced CNV complexes were quantified. Gelatin proteolysis solution assays and zymography in SDS-PAGE were performed with purified human recombinant MMP-2 and MMP-9 in the presence of doxycycline. In situ DQ-gelatin zymography was performed in retina/RPE/choroid histology sections. We found that doxycycline caused a decrease in CNV lesion volume;and that doxycycline inhibited MMP-2 and MMP-9 catalytic activities in vitro and gelatinase activities at the site of laser injury. We continued examining the effects of PEDF polypeptide fragments on vessel sprouting and on experimental CNV following subconjunctival administration in rats. Ex vivo and in vivo angiogenesis assays in the presence of recombinant human PEDF cleaved at its serpin exposed loop and synthetic PEDF peptides were evaluated. PEDF immunostaining was performed on laser-induced CNV lesions of RPE/retina sections from rats. We found that the PEDF immunoreactive signal diminished after laser injury, and that optimal range dosages of rhuPEDF or 34-mer inhibited angiogenesis, suppressed and regressed CNV lesions. We continued evaluating potential matrixes for ocular delivery devices for PEDF. Fluorescein conjugated ovalbumin, a serpin closely related to PEDF, was mixed with a biodegradable, biocompatible thermal-gel and injected in the subconjunctival space of rats in collaboration with the laboratory of Dr. Robert Lutz (DDKR, NIBIB). Protein diffusion toward the choroid/RPE and retina was assessed. The antiangiogenic effects of the PEDF-loaded gel were explored in laser-induced CNV complexes in rats. PEDF has binding affinity for PEDF receptor (PEDF-R) and stimulates its phospholipase A activity to liberate fatty acids from phospholipids. As docosahexaenoic acid (DHA) is the most abundant fatty acid in the retina, we continued evaluating the effects of DHA on experimental CNV in rodents in collaboration with the laboratories of Drs. Emily Chew (NEI, DECR), Norman Salem (NIAAA) and Joseph Hibbeln (NIAAA). A transgenic mouse model, engineered to carry a fat-1 gene from C. elegans, and dietary models with different amounts of omega-3 and omega-6 fatty acids were used to induce experimental CNV, and CNV lesion volumes were evaluated. The effects of DHA on proliferation, migration and tube formation of endothelial cells, as well as in migration of RPE cells in culture were assessed. In collaboration with the laboratory of Benjamin Sredni (Israel), we evaluated the effects of AS101 and SAS in experimental CNV in rats, and in vitro using monkey RPE cells in culture. The compounds were administered intraperitoneally, orally in drinking water, and as eye drops in rats. RPE cell cultures were treated with the compounds. We determined the cell viability and the levels of secreted PEDF and VEGF to the media. In collaboration with the laboratory of Dr. Nussenblatt (NEI), the effects of PEDF on murine macrophages, specifically on nitric oxide (nitrate) and cytokine production upon activation, were analyzed.
| 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 |
