carbocyanines and Macular-Degeneration

Cell replacement has the potential to be applied as a therapeutic strategy in retinal degenerative diseases such as retinitis pigmentosa and age-related macular degeneration (AMD) for which no adequate pharmacological and surgical treatments are currently available. Although controversial, the use of ciliary epithelium (CE)-derived cells is supported by evidence showing their differentiation into retinal phenotypes. This study examines the differentiation potential of porcine CE-derived cells in vitro and their survival, migration, morphological characteristics, and immunohistochemical phenotype in vivo, upon transplantation into the subretinal space of normal pigs.. Cells were isolated from the CE of postnatal pigs and were grown in a suspension sphere culture. Differentiation was assessed in vitro after exposure to laminin and the addition of serum. For transplantation, CE-derived spheres were dissociated, labeled with CM-DiI vital dye, and the cells were injected subretinally into one eye of eight week-old allorecipients. The eyes were examined at eight days and at two and four weeks after transplantation.. Cells positive for neuronal and retinal pigment epithelium (RPE) markers were detected by immunohistochemistry in differentiation cultures. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) revealed upregulation of neuronal markers after in vitro differentiation. CM-DiI dye-labeled CE-derived cells dissociated from primary spheres survived for up to four weeks after transplantation in vivo. Some of the surviving cells migrated distantly from the injection site. Large clusters of transplanted cells integrated into the RPE layer and multilayered RPE-like structures positive for RPE65 were often observed. Grafted cells were also identified in the neuroretina where 5%-10% were positive for recoverin, protein kinase C alpha (PKCα), and calbindin.. The efficient conversion to an RPE-like phenotype suggests that CE-derived cells could be a potential source of RPE for cell replacement. Our data also suggest that the ability of these cells to acquire neuronal phenotypes is influenced by the environment. Thus, pre-differentiated or (re)programmed CE-derived cells may be more amenable for retinal repair.

Topics: Animals; Animals, Newborn; Biomarkers; Carbocyanines; Carrier Proteins; Cell Differentiation; Cell Transplantation; Cells, Cultured; cis-trans-Isomerases; Epithelial Cells; Eye Proteins; Fluorescent Dyes; Immunohistochemistry; Injections, Intraocular; Laminin; Macular Degeneration; Neurons; Retinal Pigment Epithelium; Retinitis Pigmentosa; Reverse Transcriptase Polymerase Chain Reaction; Swine; Transplantation, Homologous

To measure changes in nuclear gene expression resulting from mitochondrial dysfunction in retinal pigment epithelial cells.. ARPE-19 retinal pigment epithelial cells were depleted of their mitochondrial (mt)DNA by passaging in a low concentration of ethidium bromide. Loss of mitochondrial DNA was determined by uridine auxotrophy and quantitative real-time polymerase chain reaction of isolated DNA. Loss of mitochondrial membrane potential was estimated by uptake of JC-1. Changes in nuclear gene expression were determined by quantitative real-time reverse transcription-polymerase chain reaction of isolated total RNA from ethidium-bromide-treated and untreated cells. Morphologic and phenotypic changes were determined by phase-contrast microscopy, sensitivity to the oxidant tert-butyl hydroperoxide (tBH), and invasion assay.. ARPE-19 cells became auxotrophic for growth on uridine after eight passages in 50 ng/mL ethidium bromide. Quantitative PCR revealed almost complete loss of mitochondrial DNA (rho(0) cells). Uptake of JC-1 was reduced in the rho(0) cells, indicating reduction of mitochondrial membrane potential. Quantitative RT-PCR measured increased expression of genes coding for drusen components, lipid transport, extracellular matrix components, and responses to inflammation in the rho(0) cells. The rho(0) cells also exhibited an increased sensitivity to killing by tBH and increased migration and invasion through solubulized basement membrane-coated tissue culture inserts.. ARPE-19 cells respond to loss of mitochondrial function by changes in nuclear gene expression that resemble changes observed in age-related macular degeneration. The results lead to the hypothesis that loss of mitochondrial function with age and resultant changes in nuclear gene expression may explain some of the changes in the macula that are associated with the known clinical manifestations of age-related macular degeneration.

Topics: Benzimidazoles; Carbocyanines; Cell Survival; Cells, Cultured; DNA, Mitochondrial; Down-Regulation; Ethidium; Fluorescent Dyes; Gene Expression Regulation; Humans; Macular Degeneration; Membrane Potentials; Mitochondrial Diseases; Nuclear Proteins; Pigment Epithelium of Eye; Reverse Transcriptase Polymerase Chain Reaction; tert-Butylhydroperoxide