Extracellular matrix nitration alters growth factor release and activates bioactive complement in human retinal pigment epithelial cells.

Purpose: We have shown previously that non-enzymatic nitration (NEN) of the extracellular matrix (ECM), which serves as a model of Bruch's membrane (BM) aging, has a profound effect on the behavior of the overlying retinal pigment epithelial (RPE) cells, including altered phagocytic ability, reduced cell adhesion, and inhibition of proliferation. We know that transplanted RPE monolayers will encounter a hostile sub-RPE environment, including age-related alterations in BM that may compromise cell function and survival. Here we use our previous NEN model of BM aging to determine the effects of NEN of the ECM on growth factor release and complement activation in RPE cells.

Differentiation of Human Protein-Induced Pluripotent Stem Cells toward a Retinal Pigment Epithelial Cell Fate.

Compared with many induced pluripotent stem cell (iPSC) lines generated using retrovirus and other non-integrating methods, the utilization of human protein-induced iPSC (piPSC) lines may provide a safer alternative for the generation of retinal pigment epithelial (RPE) cells for transplantation in retinal degenerative diseases. Here we assess the ability of piPSCs to differentiate into RPE cells, and to perform native RPE cell behavior. piPSCs were seeded in 6-well low-attachment plates to allow embryoid body formation, and then analyzed for pluripotent stem cell markers NANOG, SSEA4 and TRA-1-60 by immunofluorescence.

Reengineering Human Bruch's Membrane Increases Rod Outer Segment Phagocytosis by Human Retinal Pigment Epithelium.

Purpose: We have shown previously that Bruch's membrane (BM) aging decreases retinal pigment epithelium (RPE) phagocytosis. Herein, we determine the effects of BM reengineering on RPE phagocytosis.

Methods: BM explants were dissected from young and old donor eyes.

Nitrite Modification of Extracellular Matrix Alters CD46 Expression and VEGF Release in Human Retinal Pigment Epithelium.

Purpose: Loss of CD46 has recently been implicated in choroidal neovascularization in mice. Herein we investigated the effect of nitrite modification of the extracellular matrix (ECM) as an in vitro model of "aging" and its effect on CD46 expression and vascular endothelial growth factor (VEGF) release in cocultured human retinal pigment epithelium (RPE).

Methods: ARPE-19 cells were plated onto RPE-derived ECM conditions (untreated; nitrite modified; nitrite modified followed by washing with Triton X-100; or nitrite modified followed by washing with Triton X-100 and coated with extracellular matrix ligands).

7-Ketocholesterol is present in lipid deposits in the primate retina: potential implication in the induction of VEGF and CNV formation.

Purpose: 7-Ketocholesterol is a highly toxic oxysterol found in abundance in atherosclerotic plaques and is believed to play a critical role in atherosclerosis. The purpose of this study was to identify and localize 7-ketocholesterol (7kCh) in the primate retina and to examine the potential consequences of its presence in oxidized lipid deposits in the retina.

Methods: Unsterified 7kCh was identified and quantified by high-performance liquid chromatography-mass spectrometry.

Peroxiredoxin 3 (PDRX3) is highly expressed in the primate retina especially in blue cones.

Oxidative stress and loss of mitochondrial function have been implicated in age-related ocular diseases and thus studying enzymes involved in these processes may be of particular importance in these diseases. Peroxiredoxin III (PRDX3) is one of a family of six known peroxiredoxins which are known to protect cells against oxidative damage. PRDX3 is localized to the mitochondria and has been reported to be induced by hydrogen peroxide in aortic endothelial and lens epithelial cells.

Molecular dynamics of photoreceptor synapse formation in the developing chick retina.

The cellular and molecular mechanisms underlying photoreceptor synaptogenesis are poorly understood. Furthermore, a detailed picture of the molecular composition of photoreceptor synapses, or their subtypes, is not yet available, nor do we know what differences, if any, exist among those subtypes. To address these questions, we investigated temporal and spatial patterns of expression and assembly of photoreceptor presynaptic components during chick embryo retinal development and early posthatched life by using reverse transcriptase polymerase chain reaction (RT-PCR), dissociated retinal cells, laser-capture microdissection (LCM), immunocytochemistry and confocal microscopy.

Effects of follistatin overexpression on cell differentiation in the chick embryo retina.

Although activin is expressed in the embryonic central nervous system (CNS), its possible functions in the regulation of CNS neuronal differentiation remain largely unknown. We have investigated this question in the retina, a well-characterized CNS structure previously shown to respond to activin in vitro, and to express activin subunits and receptors in vivo. RCAS retroviruses were used to overexpress in the chick retina in ovo either follistatin (FS), an activin-binding protein and inhibitor, or alkaline phosphatase (AP), as control.