We continued studying the effects of doxycycline (bacteriostatic antibiotic with anti-metalloproteolytic and antiangiogenic properties) on CNV. In collaboration with the laboratory of Dr. Robert Fariss (NEI, BIC) we administered doxycycline orally to rats and measured plasma levels of PEDF, matrix metalloproteinases MMP-2 and MMP-9 proteins by ELISA. We induced experimental CNV and then administered doxycycline orally to rats. Using the imaging technique developed by our group for the evaluation and quantification of laser-induced choroidal neovascularization (CNV) complexes in rodents we quantified the CNV complex volumes. We found that doxycycline caused a decrease in CNV lesion volume. 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 inhibited MMP-2 and MMP-9 catalytic activities in vitro and gelatinase activities at the site of laser injury.? ? We continued the studies on the effects of extracellular matrix degrading enzymes on PEDF in collaboration with the laboratory of Dr. William Stetler-Stevenson (NCI). The effects of pH on the MMP-2 enzyme and the effects of phosphate/calcium buffers on PEDF and MMP-2 were investigated. We found that MMP-2 self proteolyzes at an optimal pH of 5.5 - 6 and that PEDF and MMP-2 precipitated in phosphate/Ca buffer. ? ? We continued investigating a biodegradable, biocompatible thermal-gelling system as potential matrix for ocular delivery devices. Fluorescein conjugated ovalbumin, a serpin closely related to PEDF, was mixed with the 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 evaluated. A similar approach using PEDF protein was used to examine the functional effects of the system. Using the imaging technique developed by our group for the evaluation and quantification of laser-induced CNV complexes in rodents, the antiangiogenic effects of the diffused PEDF were explored. ? ? PEDF has binding affinity for PEDF receptor (PEDF-R) and stimulates its phospholipase A activity that liberates fatty acids from phospholipids. Being docosahexaenoic acid (DHA) 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). We utilized a transgenic mouse model, engineered to carry a fat-1 gene from C. elegans, which results in an enrichment of omega-3 fatty acids, in particular in DHA in the brain and retina. Dietary models with different amounts of omega-3 and omega-6 fatty acids were also used. Experimental CNV was induced in these animals and CNV lesion volumes were measured. We also tested the effect of DHA on proliferation, migration and tube formation of endothelial cells as well as in migration of RPE cells in culture.
| Campos, Mercedes; Amaral, Juan; Becerra, S Patricia et al. (2006) A novel imaging technique for experimental choroidal neovascularization. Invest Ophthalmol Vis Sci 47:5163-70 |
| Amaral, Juan; Fariss, Robert N; Campos, Maria M et al. (2005) Transscleral-RPE permeability of PEDF and ovalbumin proteins: implications for subconjunctival protein delivery. Invest Ophthalmol Vis Sci 46:4383-92 |
| Notari, Luigi; Miller, Amanda; Martinez, Alfredo et al. (2005) Pigment epithelium-derived factor is a substrate for matrix metalloproteinase type 2 and type 9: implications for downregulation in hypoxia. Invest Ophthalmol Vis Sci 46:2736-47 |
