transforming-growth-factor-beta and Retinal-Degeneration

Excessive light exposure can damage photoreceptors and lead to blindness. Oxidative stress serves a key role in photo-induced retinal damage. Free radical scavengers have been proven to protect against photo-damaged retinal degeneration. Fullerol, a potent antioxidant, has the potential to protect against ultraviolet-B (UVB)-induced cornea injury by activating the endogenous stem cells. However, its effects on cell fate determination of Müller glia (MG) between gliosis and de-differentiation remain unclear. Therefore, we established a MG lineage-tracing mouse model of light-induced retinal damage to examine the therapeutic effects of fullerol. Fullerol exhibited superior protection against light-induced retinal injury compared to glutathione (GSH) and reduced oxidative stress levels, inhibited gliosis by suppressing the TGF-β pathway, and enhanced the de-differentiation of MG cells. RNA sequencing revealed that transcription candidate pathways, including Nrf2 and Wnt10a pathways, were involved in fullerol-induced neuroprotection. Fullerol-mediated transcriptional changes were validated by qPCR, Western blotting, and immunostaining using mouse retinas and human-derived Müller cell lines MIO-M1 cells, confirming that fullerol possibly modulated the Nrf2, Wnt10a, and TGF-β pathways in MG, which suppressed gliosis and promoted the de-differentiation of MG in light-induced retinal degeneration, indicating its potential in treating retinal diseases.

Topics: Animals; Ependymoglial Cells; Gliosis; Humans; Mice; Neuroglia; NF-E2-Related Factor 2; Retina; Retinal Degeneration; Transforming Growth Factor beta

Transforming growth factor β (TGFβ) signaling has manifold functions such as regulation of cell growth, differentiation, migration, and apoptosis. Moreover, there is increasing evidence that it also acts in a neuroprotective manner. We recently showed that TGFβ receptor type 2 (

Topics: Animals; Disease Models, Animal; Ependymoglial Cells; Mice; Mitogen-Activated Protein Kinases; Receptor, Transforming Growth Factor-beta Type II; Retina; Retinal Degeneration; Transforming Growth Factor beta

Müller cells may have stem cell-like capability as they regenerate photoreceptor loss upon injury in some vertebrates, but not in mammals. Indeed, mammalian Müller cells undergo major cellular and molecular changes summarized as reactive gliosis. Transforming growth factor beta (TGFβ) isoforms are multifunctional cytokines that play a central role, both in wound healing and in tissue repair. Here, we studied the role of TGFβ isoforms and their signaling pathways in response to injury induction during tissue regeneration in zebrafish and scar formation in mouse. Our transcriptome analysis showed a different activation of canonical and non-canonical signaling pathways and how they shaped the injury response. In particular, TGFβ3 promotes retinal regeneration via Smad-dependent canonical pathway upon regulation of junb gene family and mycb in zebrafish Müller cells. However, in mice, TGFβ1 and TGFβ2 evoke the p38MAPK signaling pathway. The activation of this non-canonical pathway leads to retinal gliosis. Thus, the regenerative versus reparative effect of the TGFβ pathway observed may rely on the activation of different signaling cascades. This provides one explanation of the different injury response in zebrafish and mouse retina.

Topics: Animals; Ependymoglial Cells; Fibrinolysis; Fibrosis; Gliosis; Green Fluorescent Proteins; Kinetics; Lasers; MAP Kinase Signaling System; Mice, Transgenic; Plasminogen Activator Inhibitor 1; Protein Isoforms; Regeneration; Retinal Degeneration; Signal Transduction; Tomography, Optical Coherence; Transforming Growth Factor beta; Transforming Growth Factor beta2; Up-Regulation; Zebrafish

Growth differentiation factor 15 (GDF15) is a protein belonging to the transforming growth factor beta (TGF-β) superfamily. The precursor GDF15 is cleaved and activated as a mature GDF15 by protease. GDF15 has been detected in the aqueous humor of the primary open angle glaucoma patients, however the localization and the effect on the retinal ganglion cells (RGCs) are still unknown. Thus, the purpose of this study was to elucidate the effect of GDF15 on mouse optic nerve crush (ONC) model and primary culture of rat RGCs. Immunostaining showed that the GDF15 was in the ganglion cell layer (GCL), and colocalized with GFAP-positive cells in the GCL and the optic nerve. Western blotting analysis showed that the mature GDF15 was upregulated in the retina and the optic nerve after the ONC. Intravitreal injection of GDF15 suppressed RGCs loss of the ONC model mice in vivo. The neurites length of the primary culture of rat RGCs were increased by mature GDF15 treatment. In addition, the neurotrophic effect of GDF15 was canceled by RET inhibitor treatment. These findings indicate that GDF15 has neuroprotective effect on RGCs via GFRAL-RET pathway. Therefore, GDF15 may be one of novel therapeutic targets in RGC degenerative diseases.

Topics: Animals; Blotting, Western; Cells, Cultured; Disease Models, Animal; Glial Fibrillary Acidic Protein; Growth Differentiation Factor 15; Immunohistochemistry; Male; Mice; Microscopy, Confocal; Nerve Crush; Optic Nerve Injuries; Rats; Rats, Sprague-Dawley; Retinal Degeneration; Retinal Ganglion Cells; Tissue Distribution; Transforming Growth Factor beta

Hereditary retinal degenerations like retinitis pigmentosa (RP) are among the leading causes of blindness in younger patients. To enable in vivo investigation of cellular and molecular mechanisms responsible for photoreceptor cell death and to allow testing of therapeutic strategies that could prevent retinal degeneration, animal models have been created. In this study, we deeply characterized the transcriptional profile of mice carrying the transgene rhodopsin V20G/P23H/P27L (VPP), which is a model for autosomal dominant RP. We examined the degree of photoreceptor degeneration and studied the impact of the VPP transgene-induced retinal degeneration on the transcriptome level of the retina using next generation RNA sequencing (RNASeq) analyses followed by weighted correlation network analysis (WGCNA). We furthermore identified cellular subpopulations responsible for some of the observed dysregulations using in situ hybridizations, immunofluorescence staining, and 3D reconstruction. Using RNASeq analysis, we identified 9256 dysregulated genes and six significantly associated gene modules in the subsequently performed WGCNA. Gene ontology enrichment showed, among others, dysregulation of genes involved in TGF-β regulated extracellular matrix organization, the (ocular) immune system/response, and cellular homeostasis. Moreover, heatmaps confirmed clustering of significantly dysregulated genes coding for components of the TGF-β, G-protein activated, and VEGF signaling pathway. 3D reconstructions of immunostained/in situ hybridized sections revealed retinal neurons and Müller cells as the major cellular population expressing representative components of these signaling pathways. The predominant effect of VPP-induced photoreceptor degeneration pointed towards induction of neuroinflammation and the upregulation of neuroprotective pathways like TGF-β, G-protein activated, and VEGF signaling. Thus, modulation of these processes and signaling pathways might represent new therapeutic options to delay the degeneration of photoreceptors in diseases like RP.

Topics: Animals; Chemokine CCL2; Female; Gene Expression Profiling; Gene Regulatory Networks; Glial Fibrillary Acidic Protein; GTP-Binding Proteins; Male; Mice; Mice, Transgenic; Neuroglia; Neuroprotection; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta; Up-Regulation; Vascular Endothelial Growth Factor A

Constitutive TGFβ signaling is important in maintaining retinal neurons and blood vessels and is a factor contributing to the risk for age-related macular degeneration (AMD), a retinal disease involving neurodegeneration and microglial activation. How TGFβ signaling to microglia influences pathological retinal neuroinflammation is unclear. We discovered that ablation of the TGFβ receptor, TGFBR2, in retinal microglia of adult mice induced abnormal microglial numbers, distribution, morphology, and activation status, and promoted a pathological microglial gene expression profile. TGFBR2-deficient retinal microglia induced secondary gliotic changes in Müller cells, neuronal apoptosis, and decreased light-evoked retinal function reflecting abnormal synaptic transmission. While retinal vasculature was unaffected, TGFBR2-deficient microglia demonstrated exaggerated responses to laser-induced injury that was associated with increased choroidal neovascularization, a hallmark of advanced exudative AMD. These findings demonstrate that deficiencies in TGFβ-mediated microglial regulation can drive neuroinflammatory contributions to AMD-related neurodegeneration and neovascularization, highlighting TGFβ signaling as a potential therapeutic target.

Topics: Animals; Apoptosis; Choroidal Neovascularization; Ependymoglial Cells; Macular Degeneration; Mice, Knockout; Mice, Transgenic; Microglia; Receptor, Transforming Growth Factor-beta Type II; Retina; Retinal Degeneration; Signal Transduction; Synaptic Transmission; Transforming Growth Factor beta

The purpose of this study was to explore retina-intrinsic neuroinflammatory reactions, effects on neuronal survival, relationship with classic gliosis, and possible role of the toll-like receptor 4 (TLR4). To isolate the adult retina from the systemic immune system, a previously described large animal explant culture model was used in which full-thickness porcine retinal sheets can be kept in vitro for extended time periods. Explants were kept for 5 days in vitro (DIV) and were treated with either; lipopolysaccharide (LPS), a Toll-like receptor-4 (TLR4) inhibitor (CLI-095), LPS + CLI-095, or solvent vehicle throughout the culture period after which retinal sections were examined with hematoxylin and eosin staining and extensive immunohistochemistry. In addition, culture medium from all explant groups was assayed for a panel of cytokines at 2 and 5DIV. Compared with in vivo controls, vehicle controls (CT) as well as CLI-095 explants displayed moderate reduction of total thickness and number of retinal neurons with upregulation of glial fibrillary acidic protein (GFAP) throughout the Müller cells. In contrast, LPS and LPS + CLI-095 treated counterparts showed extensive overall thinning with widespread neuronal degeneration but only minimal signs of classical Müller cell gliosis (limited upregulation of GFAP and no downregulation of glutamine synthetase (GS). These specimens also displayed a significantly increased expression of galectin-3 and TGF-beta activated kinase 1 (TAK1). Multiplex proteomic analysis of culture medium at 2DIV revealed elevated levels of IL-1β, IL-6, IL-4 and IL-12 in LPS-treated explants compared to CLI-095 and CT counterparts. LPS stimulation of the isolated adult retina results in substantial neuronal cell death despite only minimal signs of gliosis indicating a retina-intrinsic neuroinflammatory response directly related to the degenerative process. This response is characterized by early upregulation of several inflammatory related cytokines with subsequent upregulation of Galectin-3, TLR4 and TAK1. Pharmacological block of TLR4 does not attenuate neuronal loss indicating that LPS induced retinal degeneration is mediated by TLR4 independent neuroinflammatory pathways.

Topics: Animals; Cell Survival; Cells, Cultured; Cytokines; Disease Models, Animal; Galectin 3; Glial Fibrillary Acidic Protein; Gliosis; Glutamate-Ammonia Ligase; Inflammation; Lipopolysaccharides; Nerve Degeneration; Proteomics; Retinal Degeneration; Retinal Neurons; Swine; Toll-Like Receptor 4; Transforming Growth Factor beta

In contrast to the mammalian retina, the zebrafish retina exhibits the potential for lifelong retinal neurogenesis and regeneration even after severe damage. Previous studies have shown that the transforming growth factor beta (TGFβ) signaling pathway is activated during the regeneration of different tissues in the zebrafish and is needed for regeneration in the heart and the fin. In this study, we have investigated the role of the TGFβ pathway in the N-methyl-N-nitrosourea (MNU)-induced chemical model of rod photoreceptor de- and regeneration in adult zebrafish. Immunohistochemical staining for phosphorylated Smad3 was elevated during retinal regeneration, and phosphorylated Smad3 co-localized with proliferating cell nuclear antigen and glutamine synthetase, indicating TGFβ pathway activation in proliferating Müller glia. Inhibiting the TGFβ signaling pathway using a small molecule inhibitor (SB431542) resulted in accelerated recovery from retinal degeneration. Accordingly, we observed increased cell proliferation in the outer nuclear layer at days 3 to 8 after MNU treatment. In contrast to the observations in the heart and the fin, the inhibition of the TGFβ signaling pathway resulted in increased proliferation after the induction of retinal degeneration. A better understanding of the underlying pathways with the possibility to boost retinal regeneration in adult zebrafish may potentially help to stimulate such proliferation also in other species.

Topics: Animals; Benzamides; Cell Proliferation; Dioxoles; Ependymoglial Cells; Methylnitrosourea; Regeneration; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Zebrafish; Zebrafish Proteins

Müller glia are the resident radial glia in the vertebrate retina. The response of mammalian Müller glia to retinal damage often results in a glial scar and no functional replacement of lost neurons. Adult zebrafish Müller glia, in contrast, are considered tissue-specific stem cells that can self-renew and generate neurogenic progenitors to regenerate all retinal neurons after damage. Here, we demonstrate that regulation of TGFβ signaling by the corepressors Tgif1 and Six3b is critical for the proliferative response to photoreceptor destruction in the adult zebrafish retina. When function of these corepressors is disrupted, Müller glia and their progeny proliferate less, leading to a significant reduction in photoreceptor regeneration. Tgif1 expression and regulation of TGFβ signaling are implicated in the function of several types of stem cells, but this is the first demonstration that this regulatory network is necessary for regeneration of neurons.

Topics: Animals; Animals, Genetically Modified; Cell Proliferation; Disease Models, Animal; Ependymoglial Cells; Extracellular Matrix Proteins; Eye Proteins; Gliosis; Green Fluorescent Proteins; Homeobox Protein SIX3; Homeodomain Proteins; Mutation; Nerve Regeneration; Nerve Tissue Proteins; Photic Stimulation; Retina; Retinal Degeneration; Signal Transduction; Transforming Growth Factor beta; Up-Regulation; Zebrafish; Zebrafish Proteins

Dorsal-ventral specification in the amphibian embryo is controlled by β-catenin, whose activation in all dorsal cells is dependent on maternal Wnt11. However, it remains unknown whether other maternally secreted factors contribute to β-catenin activation in the dorsal ectoderm. Here, we show that maternal Xenopus Norrin (xNorrin) promotes anterior neural tissue formation in ventralized embryos. Conversely, when xNorrin function is inhibited, early canonical Wnt signaling in the dorsal ectoderm and the early expression of the zygotic neural inducers Chordin, Noggin, and Xnr3 are severely suppressed, causing the loss of anterior structures. In addition, xNorrin potently inhibits BMP- and Nodal/Activin-related functions through direct binding to the ligands. Moreover, a subset of Norrin mutants identified in humans with Norrie disease retain Wnt activation but show defective inhibition of Nodal/Activin-related signaling in mesoderm induction, suggesting that this disinhibition causes Norrie disease. Thus, xNorrin is an unusual molecule that acts on two major signaling pathways, Wnt and TGF-β, in opposite ways and is essential for early neuroectoderm specification.

Topics: Amino Acid Sequence; Animals; Blindness; Bone Morphogenetic Protein 4; Carrier Proteins; Conserved Sequence; Embryo, Nonmammalian; Embryonic Development; Gene Expression Regulation, Developmental; Genetic Diseases, X-Linked; Humans; Ligands; Mesoderm; Molecular Sequence Data; Nervous System Diseases; Neural Plate; Protein Binding; Retinal Degeneration; Spasms, Infantile; Transforming Growth Factor beta; Wnt Proteins; Wnt Signaling Pathway; Xenopus; Xenopus Proteins

Despite ubiquitous expression of the keratoepithelin (KE) protein encoded by the transforming growth factor beta induced/beta induced gene human clone 3 (TGFBI/BIGH3) gene, corneal dystrophies are restricted to the cornea, and no other tissues are affected. We investigated the role of TGFBI/BIGH3 in Groenouw corneal dystrophies by generating transgenic mice overexpressing TGFBI/BIGH3 containing the R555W mutation.. Transgenic animals expressing the Groenouw mutation of human TGFBI/BIGH3 were generated using lentiviral vectors. The line expressed TGFBI/BIGH3 containing the R555W mutation under the control of the phosphoglycerate kinase (PGK) promoter. Expression of the transgene was monitored by Southern and western blotting and by RT-PCR. Electroretinogram analysis was performed and four mice were subjected to complete necroscopy.. Transgene expression was observed in different organs although without specific expression in the cornea. The overall morphology of the transgenic animals was not severely affected by KE overexpression. However, we observed an age-dependent retinal degeneration both functionally and histologically. Female-specific follicular hyperplasia in the spleen and increased levels of lipofuscin in the adrenal gland were also seen in transgenic animals.. Cellular degeneration in the retina of transgenic animals suggest that perturbation of the transforming growth factor beta (TGFbeta) family regulation may affect photoreceptor survival and may induce possible accelerated aging in several tissues. No corneal phenotype could be observed, probably due to the lack of transgene expression in this tissue.

Topics: Animals; Blotting, Southern; Blotting, Western; Electroretinography; Extracellular Matrix Proteins; Female; Gene Expression Regulation; Humans; Hyperplasia; Lentivirus; Male; Mice; Mice, Transgenic; Mutant Proteins; Organ Size; Organ Specificity; Phosphoglycerate Kinase; Promoter Regions, Genetic; Reproducibility of Results; Retinal Degeneration; RNA, Messenger; Spleen; Transforming Growth Factor beta; Virus Integration

The alphaA-crystallin promoter was used to target expression of bone morphogenetic protein 7 (BMP7) to lens fiber cells in transgenic mice. Surprisingly, lens-specific expression of BMP7 induced widespread apoptosis and rapid ablation of the neural retina in multiple families. Subsequent to retinal ablation, the lens bow region shifted posteriorly until lens epithelial cells completely enveloped the lens. Lens-specific expression of FGF3 was found to rescue the loss of fiber cell differentiation. Our results show that elevated BMP7 levels can induce rapid retinal degeneration accompanied by disruption of the endogenous ocular system for fiber cell induction.

Topics: Animals; Apoptosis; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Breeding; Fibroblast Growth Factor 3; Fibroblast Growth Factors; Immunohistochemistry; In Situ Hybridization; Lens, Crystalline; Mice; Mice, Transgenic; Proto-Oncogene Proteins; Retina; Retinal Degeneration; Transforming Growth Factor beta

Alterations in the ocular vasculature are associated with retinal diseases such as retinopathy of prematurity and diabetic retinopathy. Vascular endothelial growth factor (VEGF) as a potent stimulator for normal and abnormal vascular growth has been extensively studied. However, little is known about secreted factors that negatively regulate vascular growth in ocular tissues. We now report that expression of a self-activating TGFbeta1 in the ocular lens of transgenic mice results in inhibition of retinal angiogenesis followed by retinal degeneration. Transgenic TGFbeta1 can rescue the hyperplasic hyaloid tissue and reverse the corneal deficiency in TGFbeta2-null embryos. These results demonstrate that TGFbeta signaling modulates development of ocular vasculature and cornea in a dosage-dependent manner and that TGFbeta1 can substitute for TGFbeta2 in ocular tissues.

Topics: Animals; Endothelial Growth Factors; Eye; Lymphokines; Mice; Mice, Transgenic; Neovascularization, Physiologic; Receptor Protein-Tyrosine Kinases; Receptors, Growth Factor; Receptors, Vascular Endothelial Growth Factor; Retinal Degeneration; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

This study was undertaken to determine whether experimental retinal detachment produces changes in retinal localization of three isoforms of transforming growth factor beta (TGF-beta) and the type II receptor for this protein. Neural retinas of young adult cats were detached from the pigment epithelium. Survival times varied from 3 to 28 days to study the temporal course of TGF-beta localization during retinal degeneration. ELISA assay for TGF-beta1 and -beta2 was performed on samples of fluid from the vitreous chamber to determine whether active or inactive TGF-beta was present. Confocal microscopy was used to localize TGF-beta1, -beta2 and -beta3 and the type II TGF-beta receptor at the various detachment durations. Following experimental retinal detachment the levels of TGF-beta2 increased in the vitreous chamber but no changes in TGF-beta1 were detected. Levels were increased 3 days post-detachment and continued throughout the 28 day period studied. The most prominent changes in immunolocalization occurred in the TGF-beta1 and -beta2 isoforms. Increased immunolabeling was seen in Müller cells and ganglion cell bodies. Hypertrophied Müller cell processes formed periretinal membranes that were heavily labeled by the TGF-beta2 antibody. Some increased immunostaining for TGF-beta3 was observed in the ganglion cell bodies. Labeling for the TGF-beta type II receptor was seen in Müller cells, ganglion cells and the inner and outer plexiform layers in both normal and detached retinas. Changes in localization of the receptor after detachment paralleled the changes seen in TGF-beta protein localization. These results demonstrate that retinal detachment induces the synthesis and secretion of TGF-beta2. Growth factor and receptor immunolabeling were increased in Müller cells suggesting that this isoform is involved in the retinal gliotic response and may contribute to the development of proliferative vitreoretinopathy.

Topics: Animals; Blotting, Western; Cats; Enzyme-Linked Immunosorbent Assay; Microscopy, Confocal; Protein Isoforms; Receptors, Transforming Growth Factor beta; Retinal Degeneration; Retinal Detachment; Transforming Growth Factor beta; Vitreous Body

To determine the relative level and localization of bone morphogenetic protein (BMP-4 mRNA in the retina and retinal pigmented epithelium (RPE) under normal and pathologic conditions, to seek clues regarding possible functions.. Clones isolated from an RPE cDNA library were sequenced and used as probes for northern blot analysis. Expression in the retina and RPE was investigated in mouse models using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. The effect of recombinant proteins on RPE proliferation was investigated by thymidine incorporation.. Bovine clones with high homology to BMP-2 and BMP4 were isolated from a subtracted RPE cDNA library. Northern blot analysis using the clones as probes demonstrated abundant and differential expression in adult bovine RPE, but with RT-PCR and in situ hybridization, expression was also demonstrated in mouse retinal neurons. In mice with oxygen-induced ischemic retinopathy there was a striking decrease in BMP-4 mRNA in the retina within 6 hours of the onset of hypoxia that was maintained for at least 5 days. In mice with inherited photoreceptor degeneration, there was a dramatic decrease in BMP4 mRNA in retina and RPE during and after the degeneration. mRNA for the type II BMP receptor was observed in freshly isolated and cultured RPE cells, isolated retina, and freshly isolated bovine aortic endothelial cells. Thymidine incorporation in early-passage RPE cells showed a 14-fold stimulation above control with 5% serum that was decreased to 322%, 393%, and 313% in the presence of BMP-2 (10 ng/ml), BMP4 (10 ng/ml), and transforming growth factor (TGF)-,1 (2 ng/ml), respectively.. BMP-2 and BMP-4 may serve as negative growth regulators in the retina and RPE that are downregulated by injury, to allow tissue repair. Modulation of expression of the BMPs may provide a means to control the exaggerated wound repair that occurs in proliferative retinopathies.

Topics: Animals; Blotting, Northern; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Cattle; Cell Division; Cells, Cultured; Disease Models, Animal; DNA Primers; Down-Regulation; Gene Expression; Humans; In Situ Hybridization; Infant, Newborn; Mice; Mice, Inbred C57BL; Pigment Epithelium of Eye; Protein Serine-Threonine Kinases; Recombinant Proteins; Retina; Retinal Degeneration; Retinopathy of Prematurity; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta

To determine whether the capacity to induce ACAID by antigen injection into the anterior chamber is altered in animals with genetically determined retinal degeneration and increased age.. Anterior chamber-associated immune deviation (ACAID) induced by injection of ovalbumin into the anterior chamber of the eye was studied in three rodent strains with different forms of hereditary retinal degeneration (Royal College of Surgeon [RCS] rats, retinal degeneration [rd] mice, and Norrie-Disease [ND] mice) and in different age groups (age range, 1-23 months). The data were compared with those of age-matched controls. Aqueous humors of rd mice, RCS rats, and age-matched congenic controls were investigated for concentrations of transforming growth factor-beta2 (TGF-beta2) using enzyme-linked immunosorbent assay.. ACAID was readily induced in RCS rats and ND mice irrespective of amount of retinal degeneration or aging. In rd mice ACAID could be induced in young animals but not in animals more than 12 months of age. In old rd mice, loss of ACAID was accompanied by a marked reduction in total TGF-beta2 levels in aqueous humor.. Rd mice more than 1 year of age lose the capacity of the anterior chamber to support the induction of ACAID by intracameral injection of soluble protein antigen. Because loss of ACAID correlated with a decrease in TGF-beta2 concentration in aqueous humor, it is proposed that eyes of rd mice are unable to maintain an immunosuppressive microenvironment necessary for ACAID.

Topics: Aging; Animals; Anterior Chamber; Aqueous Humor; Enzyme-Linked Immunosorbent Assay; Eye Diseases, Hereditary; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Ovalbumin; Rats; Rats, Mutant Strains; Retinal Degeneration; Transforming Growth Factor beta

The inherited retinal dystrophy observed in Royal College of Surgeons (RCS) rats is a widely used model for the study of the photoreceptor degeneration that occurs in retinitis pigmentosa and macular degeneration. The visual cell degeneration is accompanied by an abnormal accumulation of microglial cells in the retina of RCS rats presenting the dystrophy. In the present study, we show that combined stimulation of RCS dystrophic retinal Müller glial (RMG) cells with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) induced the release in culture supernatants of significantly higher amounts of tumor necrosis factor (TNF) and nitric oxide (NO) compared to nondystrophic congenic controls. In contrast, the levels of TNF and NO found in the supernatants from microglial cells were not significantly different in both strains. Interestingly, as shown by thymidine incorporation, microglial cells from RCS dystrophic rats have a prominent capacity of proliferation in culture medium compared to microglia isolated from RCS non dystrophic controls. Incubation of RMG cells and microglia with the stereoselective inhibitor of NOS, NG-monomethyl-L-arginine (L-NMMA), inhibited nitrite release in LPS + IFN-gamma-stimulated RMG cells and microglia. The addition of TGF-beta with LPS + IFN-gamma clearly inhibited TNF release in supernatants from both dystrophic and control rat RMG cells and microglia. While TGF-beta significantly inhibited nitrite synthesis in RMG cells, the effect on nitrite synthesis by microglia was very low. The retinal dystrophy observed in RCS dystrophic rats could result from an abnormal reactivity of RMG and microglial cells to release TNF and NO in response to stimulants. The immunomodulatory cytokine TGF-beta and inhibitors of NOS could be negative regulators in the cytokine network and nitrite synthesis thus interfering with the development of photoreceptor cell death.

Topics: Animals; Cell Culture Techniques; Cell Division; Enzyme Inhibitors; Interferon-gamma; Lipopolysaccharides; Neuroglia; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Rats; Rats, Mutant Strains; Recombinant Proteins; Retina; Retinal Degeneration; Salmonella typhimurium; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Phagocytosis of rod outer segments (ROS) is an important function of retinal pigment epithelial (RPE) cells. Since the details of the process are not fully known, we studied effects of cytokines produced by RPE and photoreceptor cells on phagocytosis of ROS by rat RPE cells.. RPE cells were isolated and cultivated from two strains of rats: Sprague-Dawley (SD) rats with normal phagocytosis and Royal College of Surgeons (RCS) rats, which have genetic deficiencies in ROS phagocytosis. A double immunofluorescence staining technique was used to study the effects in vitro of several cytokines on phagocytosis of ROS.. We found that transforming growth factor beta-1 (TGF-beta 1) had dose-dependent effects on RPE cells of both strains of rat: at a concentration of 10 ng/ml, TGF-beta 1 significantly (p < 0.01) reduced total ROS (to 74% of control in SD rats and to 51% of control in RCS rats), reduced bound ROS (to 56% of control in SD rats and to 48% in RCS rats), and increased the ratio of ingested ROS to total ROS (to 140% in SD rats but not significantly in RCS rats). Treatment of medium with anti-TGF-beta 1 antibody before incubation of RPE cells of SD rats with TGF-beta 1 decreased the magnitude of these effects. The cytokine acidic fibroblast growth factor (aFGF, 10 ng/ml) affected RPE cells of SD rats only, decreasing ROS ingested to 56% of control and the ratio of ingested ROS to total ROS to 64% of control. We also examined effects of basic fibroblast growth factor and insulin-like growth factor. None of the cytokines tested increased ingestion of ROS by RPE cells of RCS rats.. Our results suggest that TGF-beta 1 and aFGF have roles in regulating ROS phagocytosis by normal and dystrophic RPE cells in the rat.

Topics: Animals; Culture Media; Cytokines; Female; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; In Vitro Techniques; Male; Phagocytosis; Pigment Epithelium of Eye; Rats; Rats, Mutant Strains; Rats, Sprague-Dawley; Retinal Degeneration; Rod Cell Outer Segment; Somatomedins; Transforming Growth Factor beta