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. METHODS: Human induced-pluripotent stem cells (iPSCs) were differentiated into RPE cells, and confirmed by immunohistochemistry, confocal microscopy, and polymerase chain reaction. IPSC-derived RPE cells were plated onto RPE-derived ECM under untreated or nitrite-modified conditions. Cells were cultured for 7 days and barrier function measured by transepithelial resistance (TER). Vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), and complement component C3a were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: On average nitrite-modified ECM increased VEGF release both apically and basally by 0.15 ± 0.014 ng/mL (p <0.0001) and 0.21 ± 0.022 ng/mL (p <0.0001), respectively, in iPSC-derived RPE cells. Nitrite-modified ECM increased PEDF release in iPSC-derived RPE cells apically by 0.16 ± 0.031 ng/mL (p <0.0001), but not basally (0.27 ± 0.015 vs. 0.32 ± 0.029 ng/mL, (p >0.05)). Nitrite-modified ECM increased production of C3a in iPSC-derived RPE cells by 0.52 ± 0.123 ng/mL (p <0.05). CONCLUSION: Nitrite-modified ECM increased VEGF, PEDF release, and C3a production in human iPSC-derived RPE cells. This model demonstrates changes seen in the basement membrane can lead to alterations in the cell biology of the RPE cells that may be related to the development of age-related macular degeneration.

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. Some old BM explants were reengineered by cleaning with Triton X-100 and/or coating with extracellular matrix (ECM) ligands. ARPE-19 cell-derived ECM (ARPE-ECM) modified ("aged") by sodium nitrite was subjected to similar treatments. ARPE-19 cells were then cultured to confluence onto the different surfaces. Fluorescently-labeled bovine rod outer segments (ROS) were fed to cells with or without αVß5 integrin antibody. Image acquisition and phagocytosis quantification was performed by fluorescence microscopy and ImageJ analysis. RESULTS: Cleaning old donor-derived BM with detergent does not increase the uptake of ROS, but a combination of cleaning and coating with ECM ligands significantly increases RPE phagocytosis (54.9 ± 6.2 vs. 83.5 ± 6.5 arbitrary units; P < 0.05) to levels closer to young donor BM (123.6 ± 9.9 arbitrary units). Similar effects were observed on nitrite-modified ARPE-ECM subjected to the same treatments. Incubation of αVß5 blocking antibody with ROS significantly decreased RPE phagocytosis. CONCLUSIONS: The detrimental effects of aging BM on RPE phagocytosis can be reversed by reengineering the BM surface with detergent cleaning and recoating with ECM ligands. TRANSLATION RELEVANCE: These results demonstrate that the therapeutic success of transplanted RPE cells may require, at least in part, reengineering of diseased BM to make it a more suitable environment for attachment, survival and proper functioning of the RPE.

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. Following colony formation, piPSCs were assessed for confirmation of RPE cell differentiation by staining for zonula occludens (ZO-1), bestrophin, microphthalmia-associated transcription factor (MITF) and retinal pigment epithelium specific protein-65 (RPE65). To evaluate piPSC-RPE cell phagocytic ability, adult bovine photoreceptor rod outer segments (ROS) were fed to piPSC-RPE cells, which were analyzed by fluorescent microscopy and flow cytometry. Undifferentiated piPSCs expressed all pluripotent markers assessed and formed embryoid body aggregates after 7 days. Differentiated piPSC-RPE cells expressed ZO-1, bestrophin, MITF and RPE65, typical RPE cell markers. Flow cytometry revealed robust ingestion of fluorescently-labeled ROS by piPSC-RPE cells, which was over four-times greater than that of undifferentiated piPSCs and comparable to that of an immortalized RPE cell line. Phagocytosis activity by piPSC-RPE cells was significantly reduced after the addition of anti-integrin αVß5. In conclusion, piPSCs can be differentiated toward an RPE cell fate, expressing RPE cell markers and resembling native RPE cells in behavior. These results demonstrate that piPSCs can be differentiated into RPE-like cells using a method that has an increased safety profile, a critical consideration for the development of better treatments for retinal degenerative diseases such as age-related macular degeneration (AMD).

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). Cells were cultured for 7 days and CD46 expression was analyzed by immunohistochemistry and Western blot. Additionally, CD46 short interfering RNA (siRNA) was transfected into ARPE-19 cells, and VEGF levels were determined by ELISA. Finally, in the same ECM conditions, ARPE-19 cells were challenged with normal human serum and VEGF levels determined by ELISA. RESULTS: CD46 is expressed on the basolateral surface of ARPE-19 cells on RPE-derived ECM. Nitrite modification of ECM reduced the expression of CD46 on ARPE-19 cells by 0.5-fold (P = 0.003) and increased VEGF release in ARPE-19 cells by 1.7-fold (P < 0.001). CD46 knockdown also increased release of VEGF on the apical and basal sides of ARPE-19 cells in culture by 1.3- (P = 0.012) and 1.2-fold (P = 0.017), respectively. CONCLUSIONS: Nitrite modification of the ECM decreased CD46 expression and increased the release of VEGF from ARPE-19 cells. Changes in CD46 expression may lead to changes in VEGF and play a pathologic role in the development of age-related macular degeneration.

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. Localization of 7kCh was performed by immunohistochemistry. VEGF induction was determined by qRT-PCR. Cell viability was determined by measuring cellular dehydrogenase activity. Analyses were performed using ARPE19 and human vascular endothelial cells (HMVECs). RESULTS: 7-Ketocholesterol is localized mainly to deposits in the choriocapillaris and Bruch's membrane and on the surfaces of vascular endothelial cells of the neural retina. RPE/choriocapillaris regions contained approximately four times more 7kCh than the neural retina. In ARPE19 cells and HMVECs, oxidized LDL and 7kCh induced VEGF 8- to 10-fold above controls. Hypoxia inducible factor (HIF)-1alpha levels did not increase as a result of 7kCh treatment, suggesting an HIF-independent induction pathway. Cholesterol sulfate, a liver X receptor (LXR) antagonist, had marked attenuation of the 7kCh-mediated VEGF induction. LXR-specific siRNAs also reduced VEGF induction. Inhibition of NF-kappaB with BAY 11-7082 reduced IL-8 but not VEGF induction. CONCLUSIONS: The location of 7-kCh in the retina and its induction of VEGF in cultured RPE cells and HMVECs suggest it may play a critical role in choroidal neovascularization. The pathway for VEGF induction seems to be independent of HIF-1alpha and NF-kappaB but seems to be partially regulated by LXRs.

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. Immunocytochemistry in tissue sections and dissociated cells showed many similarities and few differences in the synaptic composition of rods and cone subtypes, which, however, were found to project to different strata within the outer plexiform layer. A striking finding was the precise timetable of expression of synaptic genes and proteins during synaptogenesis. Although mRNAs for some synaptic molecules appeared as early as embryonic day (ED) 5-8 (the time of inner retina synaptogenesis), others were undetectable before the time of onset of photoreceptor synaptogenesis on ED13, including CAST, rim2, synapsin-2, syntaxin-3, synaptotagmin, glutamate receptors -1, -4, and -5, homer-1 and -2, and tenascin-R. Most synaptic proteins in photoreceptors followed a similar sequence of expression: they were negative or weakly positive before ED13, appeared in inner segments between ED13 and ED15, became subsequently detectable in perinuclear and axonal regions, and by ED18 were assembled into synaptic terminals and became undetectable in the inner segments. The identity of the signals that regulate the coordinated expression of these synaptic components remains to be investigated.

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. Using a highly specific commercially available antibody, PRDX3 was readily detected by immunoblot in monkey neural retina. Immunohistochemical analysis of human and monkey retina localized PRDX3 mainly to the photoreceptor inner segments, the outer and inner plexiform layers, and the ganglion cells. These are areas of known high mitochondrial content. In the monkey retina some of the cone inner segments were much more strongly labeled than others. Dual labeling experiments using specific anti-cone opsin antibodies determined that the high expressing cones were of the blue subtype. By contrast, in the human retina all of the cone inner segments were immunoreactive. This difference may be due to a postmortem induction of PRDX3 in the human sample. These results suggest that PRDX3 may be important in protecting photoreceptor mitochondria especially in blue cones.

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. FS-treated retinas appeared normal until ED 8, when they showed a reduction of the inner plexiform layer, accompanied by a marked decrease in the frequency of amacrine cells. The territory lacking amacrine cells showed downregulation of transcription factors necessary for amacrine cell differentiation, such as Pax6 and AP2alpha, accompanied by ectopic expression of transcription factors associated with the development of horizontal or bipolar neurons, such as Prox1, Chx10 and NeuroM. Increases in cell death were also observed in FS-treated retinas. Taken together with previous in vitro studies, our results suggest that activin is a powerful regulator of neuronal differentiation in the central nervous system.