Therapeutics for the chorioretina

National Eye Institute

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Abstract

We assessed the retinoprotective effects of PEDF peptides delivered via eye drops in animal models of human retinitis pigmentosa (RP) to generate novel therapeutics for RP patients. Mouse models rd10 (with a Pde6 gene mutation) and rd10/Serpinf1 null (rd10 lacking the Serpinf1 gene for PEDF) were used. PEDF peptides 17-mer, 17-mer(H105A) and 17-mer(R99A) were chemically synthesized. Eyedrops of AlexaFluor488-labeled-peptides were used in retinal penetration and bioavailability assays using C57Bl6/J mice. Fluorescence was detected in vivo by fundoscopy using MICRON III, and in retinal extracts using a plate reader. Fluorescently labeled peptides delivered by eyedrops penetrated the wild-type retina. Cell death was detected in vivo applying eye drops of phosphatidylserine-binding conjugate of Bis(zinc (II)-dipicolylamine) with Texas-red (PSVue) and detecting fluorescence by fundoscopy at 1-day post-administration. PSVue fluorescence was detected in the fundi of living rd10 and rd10/Serpinf1 null mice at 17-21 days of age. Peptides were delivered via eye drops daily to RP mouse models. PSVue fluorescence detected in the fundi of mice with 17-mer and 17-mer(H105A) peptides decreased, but not with 17-mer(R99A). Retinal electrophysiology and morphology were assessed by electroretinography, and by histology, immunofluorescence and confocal microscopy after whole eye enucleation and processing, respectively. Electroretinography recordings showed that 17-mer(H105A) peptide eyedrops increased the a-wave amplitude of both rd10 and rd10/Serpinf1 null mice relative to controls, being more efficient than the effect of 17-mer peptide eyedrops. Measurements of the outer nuclear layer of retinas were performed. Mutant retinas exposed to 17-mer and 17-mer(H105A) peptides had thicker outer nuclear layers, being the 17-merH105A peptide more efficient, while 17-mer(R99A) was inefficient. Quantification of immunofluorescence of antiapoptotic B-cell lymphoma 2 (BCL2) and proapoptotic BCL2 associated X (BAX2) markers in retinas cross sections revealed that both 17-mer and 17-mer(H105A) increased and decreased the photoreceptor immunofluorescence of BCL2 and BAX2 markers, respectively, in the two mutant mice. Derivatives of the 17-mer peptides were designed and synthesized and used in assays for protection of photoreceptors using the rd10 and rd10 x Serpinf1-/- mouse models. AAV-based vector for the 17-mer(H015A) were injected intravitreally in mice with Rho P23H+/- and tested for protection of photoreceptors (in collaboration with the laboratory of Valeria Marigo). Retinal cross sections from these animals were used to evaluate the distribution of apoptotic markers and cell death in mice with and without AAV-based peptide. A study to investigate the anti-inflammatory effects of PEDF in the RPE was completed. We used human ARPE-19 cells stimulated with human recombinant tumor necrosis factor-alpha (TNF-alpha) to induce overexpression of the IL6 gene for the pro-inflammatory cytokine interleukin-6 (IL-6). We found that the viability of ARPE-19 cells decreased with TNF-alpha determined by both crystal Violet and automated assays. TNF-alpha elevated the production and secretion of IL-6 protein, as measured by ELISA. To challenge the TNF-alpha-mediated stimulation of IL-6, we used recombinant human PEDF protein. PEDF recovered the TNF-alpha-mediated loss of cell viability and repressed IL-6 gene expression, as determined by RT-PCR. PEDF attenuated the IL-6 protein secretion in a dose dependent fashion, being abolished with 100 nM PEDF. To map the region that confers the IL-6 blocking effect to the PEDF polypeptide, we used chemically synthesized peptides designed from its biologically active domains, pro-death 34-mer, and pro-survival 44-mer and 17-mer(H105A), to challenge the IL-6 overproduction. The pro-survival peptides recovered the TNF-alpha-mediated cell viability loss, and inhibited IL-6 secretion, while the 34-mer did not have an effect, suggesting a role for the pro-survival domain in blocking TNF-alpha-mediated cell death and IL-6 stimulation. In another study, ARPE-19 cells were stimulated with proinflammatory cytokines interferon gamma (IFN-gamma) and Interleukin-1 beta (IL-1 beta) to induce overproduction of IL-6. Additions of IFN-gamma to ARPE-19 increased the IL-6 levels in the media and reduced cell viability relative to cultures without IFN-gamma, as determined by ELISA and crystal violet assay, respectively. PEDF at 100 nM inhibited IL-6 secretion mediated by IFN-gamma and recovered the IFN-gamma-induced loss of viability. Additions of IL-1 beta to ARPE-19 cells increased the IL-6 production in the media and slightly reduced cell viability. PEDF at 100 nM had modest mitigatory effect on IL-6 production caused by IL-1 beta. To determine IL-6 levels in retinas of animal models of retinal degeneration, proteins were extracted from dissected mouse retinas from rd10 and wildtype mice at 15-25 days old. The determined levels of IL-6 were higher in rd10 than in wildtype mouse retinas. Muller glial cells (MGCs) produce neurotrophic factors that promote survival to PRs, being one of them PEDF. We investigated the effect of the neurotrophic activity of PEDF on MGCs of the rd10 mouse model of human retinitis pigmentosa. Pure MGCs and neuron-MGCs primary cultures were prepared from retinas of wild type and rd10 mice at postnatal 2-3 days of age. Morphology and size of cells and nuclei were assessed using phase-contrast microscopy, and fluorescence microscopy using DAPI (4,6-diamidino-2-phenylindole) staining. MGCs and photoreceptors were identified by immunocytochemistry using antibodies to glutamine synthetase and cone-rod homeobox (CRX), respectively. We inspected the actin cytoskeleton of MGCs using phalloidin probe staining by fluorescence microscopy. The neuron-MGCs cultures prepared from wild type mice formed clusters of a mixture of photoreceptors, neuronal precursors, and MGCs at different stages of differentiation. In contrast, the MGCs in neuron-MGCs cultures from rd10 mice had poorly extended lamellipodia, fewer intercellular connections and less expanded clusters with overlapping cells. The rd10 MGCs in both pure and neuron-MGCs primary cultures had a disorganized actin cytoskeleton compared to their wild type counterparts. The chemically synthesized 17-mer(H105A) peptide derived from the neurotrophic domain of the PEDF protein was added to the cultures. Cell proliferation and death were determined by bromodeoxyuridine (BrdU) nucleotide incorporation, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and/or DAPI staining, respectively. Treatment of rd10 neuron-MGCs cultures with the 17-mer(H105A) peptide increased the length of the MGC lamellipodia and decreased actin fluorescence intensity in the cluster area. The 17-mer(H105A) peptide decreased neuron cell death, and increased proliferation of MGCs.