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1.
Invest Ophthalmol Vis Sci ; 61(2): 3, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32031575

RESUMO

Purpose: Retinopathy of prematurity (ROP) is a leading cause of childhood blindness. ROP occurs as a consequence of postnatal hyperoxia exposure in premature infants, resulting in vasoproliferation in the retina. The tetraspan protein epithelial membrane protein-2 (EMP2) is highly expressed in the retinal pigment epithelium (RPE) in adults, and it controls vascular endothelial growth factor (VEGF) production in the ARPE-19 cell line. We, therefore, hypothesized that Emp2 knockout (Emp2 KO) protects against neovascularization in murine oxygen-induced retinopathy (OIR). Methods: Eyes were obtained from wildtype (WT) and Emp2 KO mouse pups at P7, P12, P17, and P21 after normoxia or hyperoxia (P7-P12) exposure. Following hyperoxia exposure, RNA sequencing was performed using the retina/choroid layers obtained from WT and Emp2 KO at P17. Retinal sections from P7, P12, P17, and P21 were evaluated for Emp2, hypoxia-inducible factor 1α (Hif1α), and VEGF expression. Whole mount images were generated to assess vaso-obliteration at P12 and neovascularization at P17. Results: Emp2 KO OIR mice demonstrated a decrease in pathologic neovascularization at P17 compared with WT OIR mice through evaluation of retinal vascular whole mount images. This protection was accompanied by a decrease in Hif1α at P12 and VEGFA expression at P17 in Emp2 KO animals compared with the WT animals in OIR conditions. Collectively, our results suggest that EMP2 enhances the effects of neovascularization through modulation of angiogenic signaling. Conclusions: The protection of Emp2 KO mice against pathologic neovascularization through attenuation of HIF and VEGF upregulation in OIR suggests that hypoxia-induced upregulation of EMP2 expression in the neuroretina modulates HIF-mediated neuroretinal VEGF expression.


Assuntos
Glicoproteínas de Membrana/fisiologia , Neovascularização Retiniana/patologia , Retinopatia da Prematuridade/patologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Animais Recém-Nascidos , Linhagem Celular , Hiperóxia/fisiopatologia , Hipóxia/fisiopatologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/patologia , Oxigênio/toxicidade , Epitélio Pigmentado da Retina/metabolismo , Vasos Retinianos/patologia , Regulação para Cima/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
2.
Cell Physiol Biochem ; 54(2): 161-179, 2020 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-32045141

RESUMO

BACKGROUND/AIMS: We performed co-culture experiments between human RPE cells (ARPE-19) and human umbilical vascular endothelial cells (HUVEC) in order to evaluate how anti-VEGF drugs could affect NO release, mitochondrial function, the oxidative status, proliferation and migration of RPE cells through modulation of their cross talk with vascular endothelial cells. METHODS: The co-culture HUVEC/RPE, was exposed to Ranibizumab/Aflibercept in the absence/presence of the NO synthase (NOS) inhibitor, the phosphatidylinositol 3'-kinase (PI3K), the extracellular-signal-regulated kinases 1/2 (ERK1/2) and the p38 mitogen-activated protein kinase (p38 MAPK) blockers. Specific kits were used for cell viability, mitochondrial membrane potential, NO, ROS and GSH production. Western blot was performed for apoptosis markers, NOS isoforms, and others kinases detection. Cell migration was analyzed by scratch assay, whereas cell proliferation and cell cycle through xCELLigence and flow cytometry. RESULTS: In RPE cells co-cultured with HUVEC in physiological conditions, Aflibercept/Ranibizumab increased NO release in a dose and time-dependent way. Opposite results were obtained in peroxidative conditions. Both anti-VEGF agents were able to prevent the fall of cell viability and mitochondrial membrane potential, an effect which was reduced by various inhibitors, and increased cell migration. Aflibercept/Ranibizumab counteracted the changes of apoptosis markers, NOS expression/activation, PI3K and ERK1/2 activation caused by peroxidation. These results were confirmed by cell cycle analysis. CONCLUSION: This study has shown new mechanisms at the basis of protective effects elicited by Aflibercept/Ranibizumab in RPE cells. HUVEC stimulated with Aflibercept/Ranibizumab, could release some paracrine factors that can modulate the RPE cells response in both physiologic and peroxidative conditions.


Assuntos
Comunicação Celular/efeitos dos fármacos , Ranibizumab/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Glutationa/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo
3.
PLoS One ; 15(2): e0228895, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32032388

RESUMO

BACKGROUND: Alpha-1-antitrypsin is a protein involved in avoidance of different processes that are seen in diabetic retinopathy pathogenesis. These processes include apoptosis, extracellular matrix remodeling and damage of vessel walls and capillaries. Furthermore, because of its anti-inflammatory effects, alpha-1-antitrypsin has been proposed as a possible therapeutic approach for diabetic retinopathy. Our group tested alpha-1-antitrypsin in a type 1 diabetes mouse model and observed a reduction of inflammation and retinal neurodegeneration. Thus, shedding light on the mechanism of action of alpha-1-antitrypsin at molecular level may explain how it works in the diabetic retinopathy context and show its potential for use in other retinal diseases. METHODS: In this work, we evaluated alpha-1-antitrypsin in an ARPE-19 human cell line exposed to high glucose. We explored the expression of different mediators on signaling pathways related to pro-inflammatory cytokines production, glucose metabolism, epithelial-mesenchymal transition and other proteins involved in the normal function of retinal pigment epithelium by RT-qPCR and Western Blot. RESULTS: We obtained different expression patterns for evaluated mediators altered with high glucose exposure and corrected with the use of alpha-1-antitrypsin. CONCLUSIONS: The expression profile obtained in vitro for the evaluated proteins and mRNA allowed us to explain our previous results obtained on mouse models and to hypothesize how alpha-1-antitrypsin hinder diabetic retinopathy progression on a complex network between different signaling pathways. GENERAL SIGNIFICANCE: This network helps to understand the way alpha-1-antitrypsin works in diabetic retinopathy and its scope of action.


Assuntos
Retinopatia Diabética/metabolismo , alfa 1-Antitripsina/metabolismo , alfa 1-Antitripsina/fisiologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Glucose/metabolismo , Humanos , Inflamação/metabolismo , Camundongos , NF-kappa B/metabolismo , Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/fisiologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo
4.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 51(1): 18-23, 2020 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-31950784

RESUMO

Objective: To study the expression and secretion of alternative complement pathway regulator complement factor H (CFH) in spontaneously produced or induced human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE). Methods: RPE cells were acquired by spontaneous differentiation from hESC (sdRPE), a source of hESC-RPE, according to the method used in clinical trials. RPE cells were also acquired under the induction of growth factors and small molecules for 14 d (iRPE). Acquired cells were kept culturing for 3 month for maturation. All differentiated cells(P3)were cultivated for 4-5 weeks prior to characterization with qRT-PCR and immunofluorescence. Secretion levels of CFH were investigated by ELISA. ARPE-19 cell line was served as control. Results: Both sdRPE and iRPE showed high similarity in cell morphology and the pattern of specific gene expression with human RPE. The relative CFH mRNA expression levels of both sdRPE and iRPE were significantly higher than that of ARPE-19 ( P<0.05). The CFH secretion levels of sdRPE in the 24 h-, 48 h- and 72 h-culture medium were higher than those of iRPE ( P=0.000 2); and this CFH secretion levels of both sdRPE and iRPE were higher than that of the ARPE-19 cell line ( P<0.000 1). Conclusion: Both sdRPE and iRPE derived by different differentiation methods expressed and secreted CFH, suggesting that hESC-RPE may have certain ability to regulate the alternative complement pathway.


Assuntos
Fator H do Complemento , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas , Epitélio Pigmentado da Retina , Linhagem Celular , Fator H do Complemento/genética , Via Alternativa do Complemento/genética , Humanos , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo
5.
Toxicol Lett ; 322: 77-86, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31931077

RESUMO

Failure of all-trans-retinal (atRAL) clearance contributes to retina degeneration. However, whether autophagy can be activated by excess atRAL accumulation in retinal pigment epithelial (RPE) cells is not known. This study showed that atRAL provoked mitochondria-associated reactive oxygen species (ROS) production, activated the nuclear factor (erythroid-derived 2)-like 2 and apoptosis in a human RPE cell line, ARPE-19 cells. Moreover, we found that autophagic flux was functionally activated after atRAL treatment. The antioxidant N-acetylcysteine attenuated the expression of autophagy markers, suggesting that ROS triggered atRAL-activated autophagy. In addition, autophagic cell death was observed in atRAL-treated RPE cells, while inhibition of autophagy with 3-methyladenine or LC3, Beclin1, p62 silencing ameliorated atRAL-induced cytotoxicity. Suppression of autophagy quenched mitochondrial ROS and inhibited HO-1 and γ-GCSh expression, indicating that atRAL-activated autophagy enhances intracellular oxidative stress, thereby promoting RPE cell apoptosis. Furthermore, we found that inhibiting endoplasmic reticulum (ER) stress suppressed atRAL-induced mitochondrial ROS generation, subsequently attenuated autophagy and apoptosis in RPE cells. Taken together, these results suggest that atRAL-induced oxidative stress and ER stress modulate autophagy, which may contribute to RPE degeneration. There may be positive feedback regulatory mechanisms between atRAL-induced oxidative stress and autophagy or ER stress.


Assuntos
Autofagia/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Vitamina A/toxicidade , Apoptose/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Transdução de Sinais
6.
Invest Ophthalmol Vis Sci ; 60(15): 5059-5069, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31800964

RESUMO

Purpose: Beta-adrenergic receptor (AR) antagonists, like propranolol, inhibit angiogenesis in multiple ocular conditions through an unknown mechanism. We previously showed that propranolol reduces choroidal neovascularization (CNV) by decreasing interleukin-6 levels. Since macrophages are one of the central producers of interleukin-6, we examined whether macrophages are required for propranolol-driven inhibition of choroidal angiogenesis. Methods: We tested the anti-angiogenic properties of propranolol in the choroidal sprouting assay and the laser-induced CNV model. Bone marrow-derived monocytes (BMDMs) were added to the choroidal sprouting assay and Ccr2-/- mice were subjected to laser-induced CNV. Multi-parameter flow cytometry was performed to characterize the ocular mononuclear phagocyte populations after laser injury and during propranolol treatment. Results: Propranolol reduced choroidal angiogenesis by 41% (P < 0.001) in the choroidal sprouting assay. Similarly, propranolol decreased laser-induced CNV by 50% (P < 0.05) in female mice, with no change in males. BMDMs increased choroidal sprouting by 146% (P < 0.0001), and this effect was ablated by propranolol. Beta-AR inhibition had no effect upon laser-induced CNV area in female Ccr2-/- mice. MHCII+ and MHCII- macrophages increased 20-fold following laser treatment in wildtype mice as compared to untreated mice, and this effect was completely attenuated in lasered Ccr2-/- mice. Moreover, propranolol increased the numbers of MHCII+ and MHCII- macrophages by 1.9 (P = 0.07) and 3.1 (P < 0.05) fold in lasered female mice with no change in macrophage numbers in males. Conclusions: Our data suggest that propranolol inhibits angiogenesis through recruitment of monocyte-derived macrophages in female mice only. These data show the anti-angiogenic nature of beta-AR blocker-recruited monocyte-derived macrophages in CNV.


Assuntos
Neovascularização de Coroide/tratamento farmacológico , Angiofluoresceinografia/métodos , Macrófagos/patologia , Monócitos/patologia , Propranolol/farmacologia , Receptores Adrenérgicos beta/metabolismo , Antagonistas Adrenérgicos beta/farmacologia , Animais , Corioide/metabolismo , Corioide/patologia , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Fundo de Olho , Imageamento Tridimensional , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Receptores Adrenérgicos beta/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia
7.
Invest Ophthalmol Vis Sci ; 60(15): 5022-5034, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31791063

RESUMO

Purpose: Retinal pigment epithelium (RPE) limits the xenobiotic entry from the systemic blood stream to the eye. RPE surface transporters can be important in ocular drug distribution, but it has been unclear whether they are expressed on the apical, basal, or both cellular surfaces. In this paper, we provide quantitative comparison of apical and basolateral RPE surface proteomes. Methods: We separated the apical and basolateral membranes of differentiated human fetal RPE (hfRPE) cells by combining apical membrane peeling and sucrose density gradient centrifugation. The membrane fractions were analyzed with quantitative targeted absolute proteomics (QTAP) and sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) to reveal the membrane protein localization on the RPE cell surfaces. We quantitated 15 transporters in unfractionated RPE cells and scaled their expression to tissue level. Results: Several proteins involved in visual cycle, cell adhesion, and ion and nutrient transport were expressed on the hfRPE plasma membranes. Most drug transporters showed similar abundance on both RPE surfaces, whereas large neutral amino acids transporter 1 (LAT1), p-glycoprotein (P-gp), and monocarboxylate transporter 1 (MCT1) showed modest apical enrichment. Many solute carriers (SLC) that are potential prodrug targets were present on both cellular surfaces, whereas putative sodium-coupled neutral amino acid transporter 7 (SNAT7) and riboflavin transporter (RFT3) were enriched on the basolateral and sodium- and chloride-dependent neutral and basic amino acid transporter (ATB0+) on the apical membrane. Conclusions: Comprehensive quantitative information of the RPE surface proteomes was reported for the first time. The scientific community can use the data to further increase understanding of the RPE functions. In addition, we provide insights for transporter protein localization in the human RPE and the significance for ocular pharmacokinetics.


Assuntos
Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Proteômica/métodos , Epitélio Pigmentado da Retina/metabolismo , Transporte Biológico , Western Blotting , Adesão Celular , Células Cultivadas , Humanos , Epitélio Pigmentado da Retina/embriologia
8.
Invest Ophthalmol Vis Sci ; 60(15): 5104-5111, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31826237

RESUMO

Purpose: Cell-cell contact in retinal pigment epithelium (RPE) involves adherent junctions, gap junctions, and tight junctions, which are primarily composed by E-cadherin, zona occludens 1 (ZO-1), and connexin 43, respectively. Here, we aimed to explore the relationship and interplay between these junction-associated proteins. Methods: E-cadherin, connexin 43, and ZO-1 expression in human primary RPE in the early phase after TGF-ß1 stimulation was detected. The knockdown of E-cadherin, ZO-1, and connexin 43 was performed to characterize the regulatory network involving these three proteins. Dye transfer and FITC-dextran permeability assays were conducted to observe the epithelial functional alterations. Transmission electron microscopy (TEM) was used to observe the ultrastructure of the cell-cell junctions in mouse RPE. The immunofluorescence staining and coimmunoprecipitation were performed to observe the colocalization and the physical association of E-cadherin, ZO-1, and connexin 43. Results: Among these three components, E-cadherin appeared to be the first protein that was downregulated after TGF-ß1 treatment. The ultrastructures of adherent junctions, gap junctions, and tight junctions could be observed in mouse RPE by TEM. E-cadherin, ZO-1, and connexin 43 were colocalized and physically bound to each other. The knockdown of one of these three proteins led to downregulation of the other two proteins and compromised epithelial function. Conclusions: E-cadherin, ZO-1, and connexin 43 were physically associated with each other and were mutually regulated. To enhance the understanding of cell-cell contacts, a holistic view is needed. Our results provide new insights in RPE disorders such as proliferative vitreoretinopathy.


Assuntos
Caderinas/genética , Conexina 43/genética , Regulação da Expressão Gênica , Epitélio Pigmentado da Retina/metabolismo , Vitreorretinopatia Proliferativa/genética , Proteína da Zônula de Oclusão-1/genética , Animais , Caderinas/biossíntese , Células Cultivadas , Conexina 43/biossíntese , Humanos , Junções Intercelulares , Camundongos , Microscopia Eletrônica de Transmissão , RNA/genética , Epitélio Pigmentado da Retina/ultraestrutura , Junções Íntimas , Vitreorretinopatia Proliferativa/metabolismo , Vitreorretinopatia Proliferativa/patologia , Proteína da Zônula de Oclusão-1/biossíntese
9.
Adv Exp Med Biol ; 1185: 21-25, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884583

RESUMO

The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier, provides nutrients, recycles visual pigment, and removes spent discs from the photoreceptors, among many other functions. Because of these critical roles in visual homeostasis, the RPE is a principal location of disease-associated changes in age-related macular degeneration (AMD), emphasizing its importance for study in both visual health and disease. Unfortunately, there are no early indicators of AMD or disease progression, a void that could be filled by the development of early AMD biomarkers. Exosomes are lipid bilayer membrane vesicles of nanoscale sizes that are released in a controlled fashion by cells and carry out a number of extra- and intercellular activities. In the RPE they are released from both the apical and basal sides, and each source has a unique signature/content. Exosomes released from the basolateral side of RPE cells enter the systemic circulation via the choroid and thus represent a potential source of retinal disease biomarkers in blood. Here we discuss the potential of targeted immunocapture of eye-derived exosomes and other small extracellular vesicles from blood for eye disease biomarker discovery.


Assuntos
Biomarcadores/sangue , Exossomos/metabolismo , Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Corioide , Humanos , Degeneração Macular/patologia
10.
Adv Exp Med Biol ; 1185: 51-55, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884588

RESUMO

The importance of cholesterol as a structural component of photoreceptors and the association between impaired cholesterol homeostasis and age-related macular degeneration (AMD) prompted in the last years a deep investigation of its metabolism in the retina. Here, we focus on the export of cholesterol from intracellular membranes to extracellular acceptors, an active mechanism mediated by the ATP-binding cassette transporters A1 and G1 (ABCA1 and G1) also known as "active cholesterol efflux." Expression of genes involved in this pathway was shown for most retinal cells, while functional in vitro assays focused on the retinal pigment epithelium (RPE) due to availability of cell models. Cell-specific knockout (KO) mice were generated in the past years, and their characterization unveils an important role of the ABCA1/G1 pathway in RPE, rods, and retinal inflammatory cells. The actual involvement of cholesterol efflux in the pathogenesis of AMD still needs to be demonstrated and will help in establishing the scientific rationale for targeting the ABCA1/G1 pathway in retinal diseases.


Assuntos
Colesterol/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico , Degeneração Macular/metabolismo , Camundongos , Camundongos Knockout
11.
Adv Exp Med Biol ; 1185: 289-293, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884626

RESUMO

The retinal pigment epithelium (RPE) is a monolayer of pigmented cells whose function is essential for the integrity of the retina and for visual function. Retinal diseases that eventually end in vision loss and blindness involve inflammation, oxidative stress (OS), and alterations in the RPE-photoreceptor cellular partnership. This chapter summarizes the role of lipid signaling pathways and lipidic molecules in RPE cells exposed to inflammatory and OS conditions. The modulation of these pathways in the RPE, through either enzyme inhibitors or receptor stimulation or blockage, could open new therapeutic strategies for retinal degenerative diseases.


Assuntos
Lipídeos/fisiologia , Estresse Oxidativo , Degeneração Retiniana/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais , Humanos , Epitélio Pigmentado da Retina/citologia
12.
Invest Ophthalmol Vis Sci ; 60(14): 4632-4642, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31682714

RESUMO

Purpose: Targeting ß-adrenergic receptor signaling with propranolol has emerged as a potential candidate to counteract choroidal neovascularization (CNV). Little is known of its effect on macrophages, which play a critical role in CNV. We investigated the effect of propranolol on angiogenic response of mononuclear phagocytes (MPs). Methods: The angiogenic effect of propranolol was evaluated in laser-induced CNV model. Mice received intraperitoneal injections of propranolol (6 mg/kg/d) or vehicle. CNV area and inflammatory cells were determined respectively by using lectin staining and an anti-IBA-1 antibody on RPE/choroid flat mounts. Inflammatory gene expression was evaluated by quantitative (q) PCR analysis. Mechanisms of propranolol was studied in MP cell lines J774 and RAW264.7 and in primary peritoneal macrophages. Expression of pro- and antiangiogenic mediators was studied. In addition, effects of propranolol treatment of MPs was assessed on choroidal explant. Results: CNV was attenuated by propranolol and concomitantly associated with decreased inflammatory mediators IL-6 and TNFα, albeit with accumulation of (ß-adrenoceptor harboring) MPs in the CNV area. Conditioned media from MPs preincubated with propranolol exerted antiangiogenic effects. Treatment of J774 confirmed the attenuation of inflammatory response to propranolol and increased cleaved caspase-3 on choroidal explant. We found that propranolol increased pigment epithelium-derived factor (PEDF) expression in MPs. Trapping of PEDF with an antibody abrogated antiangiogenic effects of propranolol. PEDF was also detected in CNV-associated MPs. Conclusions: We hereby show that propranolol confers on MPs antiangiogenic properties by increasing PEDF expression, which complements its effects on vascular tissue resulting in inhibition of choroidal vasoproliferation in inflammatory conditions. The study supports possible use of propranolol as a therapeutic modality for CNV.


Assuntos
Antagonistas Adrenérgicos beta/uso terapêutico , Inibidores da Angiogênese/uso terapêutico , Neovascularização de Coroide/prevenção & controle , Macrófagos Peritoneais/efeitos dos fármacos , Sistema Fagocitário Mononuclear/efeitos dos fármacos , Propranolol/uso terapêutico , Animais , Western Blotting , Caspase 3/metabolismo , Linhagem Celular , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Proteínas do Olho/metabolismo , Injeções Intraperitoneais , Interleucina-6/metabolismo , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sistema Fagocitário Mononuclear/metabolismo , Fatores de Crescimento Neural/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Serpinas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
13.
Invest Ophthalmol Vis Sci ; 60(14): 4652-4660, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31743940

RESUMO

Purpose: Retinal damage in ocular toxoplasmosis reflects Toxoplasma gondii-induced cell lysis and reactive inflammation. Human retinal histopathology demonstrates the presence of neutrophils, but activities of this leukocyte subset are unstudied. We conducted in vitro experiments to evaluate roles for neutrophils as retinal taxis for T. gondii and as contributors to the inflammation. Methods: Human neutrophils were isolated from peripheral blood. Migration to disease-relevant chemokines was evaluated in transwells, seeded with human retinal endothelial cells for some assays, using neutrophils infected with GT-1 strain T. gondii tachyzoites. Neutrophils were cocultured with T. gondii-infected ARPE-19 and primary human retinal pigment epithelial cells, and production of reactive oxygen species (ROS) was estimated by dihydroethidium reaction. Proteins produced by T. gondii-infected ARPE-19 cells were profiled by immunoarray, and candidate neutrophil-activating proteins were targeted with specific blocking antibody in coculture assays. Results: Infection with T. gondii arrested neutrophil migration across retinal endothelium regardless of the presence of CXCL8. Migration to CXCL1, CXCL2, and CXCL8 also was significantly inhibited in infected neutrophils. Neutrophils generated more ROS when cocultured with infected versus uninfected ARPE-19 cells and three of four primary retinal pigment epithelial cell isolates. Infected ARPE-19 cells augmented the synthesis of 12 neutrophil-activating proteins also expressed by primary retinal pigment epithelial cells. Antibody blockade of granulocyte-macrophage colony-stimulating factor, interleukin-6 (IL-6) and IL-18 significantly reduced ROS production by neutrophils cocultured with T. gondii-infected ARPE-19 cells. Conclusions: Our findings support involvement of neutrophils in retinal inflammation, but not parasite transport, in the setting of ocular toxoplasmosis.


Assuntos
Neutrófilos/fisiologia , Epitélio Pigmentado da Retina/metabolismo , Toxoplasmose Ocular/imunologia , Adulto , Linhagem Celular , Ensaios de Migração de Leucócitos , Movimento Celular/fisiologia , Quimiocinas/metabolismo , Técnicas de Cocultura , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Interleucina-18/metabolismo , Interleucina-6/metabolismo , Ativação de Neutrófilo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Epitélio Pigmentado da Retina/parasitologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Toxoplasma/fisiologia
14.
PLoS Pathog ; 15(10): e1007956, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31589653

RESUMO

We report the analysis of a complex enveloped human virus, herpes simplex virus (HSV), assembled after in vivo incorporation of bio-orthogonal methionine analogues homopropargylglycine (HPG) or azidohomoalanine (AHA). We optimised protocols for the production of virions incorporating AHA (termed HSVAHA), identifying conditions which resulted in normal yields of HSV and normal particle/pfu ratios. Moreover we show that essentially every single HSVAHA capsid-containing particle was detectable at the individual particle level by chemical ligation of azide-linked fluorochromes to AHA-containing structural proteins. This was a completely specific chemical ligation, with no capsids assembled under normal methionine-containing conditions detected in parallel. We demonstrate by quantitative mass spectrometric analysis that HSVAHA virions exhibit no qualitative or quantitative differences in the repertoires of structural proteins compared to virions assembled under normal conditions. Individual proteins and AHA incorporation sites were identified in capsid, tegument and envelope compartments, including major essential structural proteins. Finally we reveal novel aspects of entry pathways using HSVAHA and chemical fluorochrome ligation that were not apparent from conventional immunofluorescence. Since ligation targets total AHA-containing protein and peptides, our results demonstrate the presence of abundant AHA-labelled products in cytoplasmic macrodomains and tubules which no longer contain intact particles detectable by immunofluorescence. Although these do not co-localise with lysosomal markers, we propose they may represent sites of proteolytic virion processing. Analysis of HSVAHA also enabled the discrimination from primary entering from secondary assembling virions, demonstrating assembly and second round infection within 6 hrs of initial infection and dual infections of primary and secondary virus in spatially restricted cytoplasmic areas of the same cell. Together with other demonstrated applications e.g., in genome biology, lipid and protein trafficking, this work further exemplifies the utility and potential of bio-orthogonal chemistry for studies in many aspects of virus-host interactions.


Assuntos
Aminoácidos/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Epitélio Pigmentado da Retina/virologia , Proteínas Estruturais Virais/metabolismo , Montagem de Vírus , Internalização do Vírus , Proliferação de Células , Células Cultivadas , Herpes Simples/metabolismo , Humanos , Epitélio Pigmentado da Retina/metabolismo
15.
Exp Eye Res ; 189: 107828, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31589840

RESUMO

Several lines of evidence support the existence of a renin-angiotensin system (RAS) in the retina that is separated from the blood stream by the retinal pigment epithelium (RPE). Under physiological conditions, increased activity of intraretinal RAS regulates neuronal activity of the retina but patho-physiologically participates in retinal degeneration such as hypertensive or diabetic retinopathy. Interestingly, the RPE appears to be a modulator of intraretinal RAS in response to changes in systemic RAS. As increased systemic RAS activity is associated with increased sympathetic tonus, we investigated whether systemic ß-adrenergic stimulation of the RPE also modulates renin expression in the RPE. In vivo, the mouse RPE expresses the ß-adrenergic receptor subtypes 1 and 2. Staining of retina sagittal sections showed tyrosine hydroxylase positive nerve endings in the choroid indicating adrenaline/noradrenaline production sites in close proximity to the RPE. Systemic infusion of isoproterenol increased renin expression in the RPE but not in the retina. This increase was sensitive to concomitant systemic application of the angiotensin-2 receptor-type-1 blocker losartan. In vitro analysis of renin gene expression using polarized porcine RPE showed that the activity of the renin promoter can be increased by cAMP stimulation (IBMX/forskolin) but was not influenced by angiotensin-2. Thus, with the identification of the ß-adrenergic system we added a new regulator of the retinal RAS with relevance for retinal function and pathology. Furthermore, it appears that the RPE is not only a close interaction partner of the photoreceptors but also a regulator or retinal activity in general.


Assuntos
Receptores Adrenérgicos beta/biossíntese , Sistema Renina-Angiotensina/fisiologia , Epitélio Pigmentado da Retina/metabolismo , Sistema Nervoso Simpático/fisiologia , Animais , Células Cultivadas , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Renina/biossíntese , Epitélio Pigmentado da Retina/citologia , Estimulação Química
16.
Exp Eye Res ; 189: 107838, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31622617

RESUMO

As many other organs, the retina has a local renin-angiotensin-system (RAS). All main elements of the RAS are active in the retina: renin, angiotensinogen, angiotensin-converting enzymes. The functional role of the intraretinal RAS is not fully understood. So far, histological and functional analysis point to a regulation of ganglion cell activity and maybe also of bipolar cell activity, but it is not clear how RAS contributes to retinal signal processing. In contrast to local RAS in other organs, the retinal RAS is clearly separated from the systemic RAS. The angiotensin-2 (AngII)/AngI ratio in the retina is different to that in the plasma. However, it appears that the retinal pigment epithelium (RPE), that forms the outer blood/retina barrier, is a major regulator of the retinal RAS by producing renin. Interestingly, comparable to the kidney, the renin production in the RPE is under control of the angiotensin-2 receptor type-1 (AT1). AT1 localizes to the basolateral membrane of the RPE and faces the blood side of the blood/retina barrier. Increases in systemic AngII reduce renin production in the RPE and therefore decrease the intraretinal RAS activity. The relevance of the local RAS for retinal function remains unclear. Nevertheless, it is of fundamental significance to understand the pathology of systemically induced retinal diseases such as hypertension or diabetes.


Assuntos
Barreira Hematorretiniana/metabolismo , Sistema Renina-Angiotensina/fisiologia , Renina/biossíntese , Epitélio Pigmentado da Retina/metabolismo , Animais , Humanos
17.
Int J Mol Sci ; 20(20)2019 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-31614727

RESUMO

Vascular endothelial growth factor-A (VEGF) is critical for the development, growth, and survival of blood vessels. Retinal pigmented epithelial (RPE) cells are a major source of VEGF in the retina, with evidence that the extracellular matrix (ECM)-binding forms are particularly important. VEGF associates with fibronectin in the ECM to mediate distinct signals in endothelial cells that are required for full angiogenic activity. Hypoxia stimulates VEGF expression and angiogenesis; however, little is known about whether hypoxia also affects VEGF deposition within the ECM. Therefore, we investigated the role of hypoxia in modulating VEGF-ECM interactions using a primary retinal cell culture model. We found that retinal endothelial cell attachment to RPE cell layers was enhanced in cells maintained under hypoxic conditions. Furthermore, we found that agents that disrupt VEGF-fibronectin interactions inhibited endothelial cell attachment to RPE cells. We also found that hypoxia induced a general change in the chemical structure of the HS produced by the RPE cells, which correlated to changes in the deposition of VEGF in the ECM, and we further identified preferential binding of VEGFR2 over VEGFR1 to VEGF laden-fibronectin matrices. Collectively, these results indicate that hypoxia-induced HS may prime fibronectin for VEGF deposition and endothelial cell recruitment by promoting VEGF-VEGFR2 interactions as a potential means to control angiogenesis in the retina and other tissues.


Assuntos
Endotélio Vascular/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Heparitina Sulfato/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Adesão Celular , Hipóxia Celular , Linhagem Celular , Células Cultivadas , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Humanos , Oxigênio/metabolismo , Ratos , Epitélio Pigmentado da Retina/citologia
18.
Int J Mol Sci ; 20(19)2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31569675

RESUMO

Age-related macular degeneration (AMD) is one of the main causes of vision impairment in the elderly. Autophagy is the process of delivery of cytoplasmic components into lysosomes for cleavage; its age-related malfunction may contribute to AMD. Here we showed that the development of AMD-like retinopathy in OXYS rats is accompanied by retinal transcriptome changes affecting genes involved in autophagy. These genes are associated with kinase activity, immune processes, and FoxO, mTOR, PI3K-AKT, MAPK, AMPK, and neurotrophin pathways at preclinical and manifestation stages, as well as vesicle transport and processes in lysosomes at the progression stage. We demonstrated a reduced response to autophagy modulation (inhibition or induction) in the OXYS retina at age 16 months: expression of genes Atg5, Atg7, Becn1, Nbr1, Map1lc3b, p62, and Gabarapl1 differed between OXYS and Wistar (control) rats. The impaired reactivity of autophagy was confirmed by a decreased number of autophagosomes under the conditions of blocked autophagosome-lysosomal fusion according to immunohistochemical analysis and transmission electron microscopy. Thus, the development of AMD signs occurs against the background of changes in the expression of autophagy-related genes and a decrease in autophagy reactivity: the ability to enhance autophagic flux in response to stress.


Assuntos
Autofagia , Degeneração Macular/etiologia , Degeneração Macular/metabolismo , Animais , Autofagia/genética , Biomarcadores , Biologia Computacional/métodos , Modelos Animais de Doenças , Suscetibilidade a Doenças , Imunofluorescência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Degeneração Macular/patologia , Ratos , Retina/metabolismo , Retina/patologia , Retina/ultraestrutura , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Epitélio Pigmentado da Retina/ultraestrutura , Transcriptoma
19.
Int J Mol Sci ; 20(19)2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31569695

RESUMO

Age-related macular degeneration (AMD) is a leading cause of blindness in the developed world. The retinal pigment epithelium (RPE) is a critical site of pathology in AMD. Oxidative stress plays a key role in the development of AMD. We generated a chimeric high-density lipoprotein (HDL), mimetic peptide named HM-10/10, with anti-oxidant properties and investigated its potential for the treatment of retinal disease using cell culture and animal models of RPE and photoreceptor (PR) degeneration. Treatment with HM-10/10 peptide prevented human fetal RPE cell death caused by tert-Butyl hydroperoxide (tBH)-induced oxidative stress and sodium iodate (NaIO3), which causes RPE atrophy and is a model of geographic atrophy in mice. We also show that HM-10/10 peptide ameliorated photoreceptor cell death and significantly improved retinal function in a mouse model of N-methyl-N-nitrosourea (MNU)-induced PR degeneration. Our results demonstrate that HM-10/10 protects RPE and retina from oxidant injury and can serve as a potential therapeutic agent for the treatment of retinal degeneration.


Assuntos
Lipoproteínas HDL/metabolismo , Peptídeos/farmacologia , Células Fotorreceptoras/efeitos dos fármacos , Células Fotorreceptoras/metabolismo , Degeneração Retiniana/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Animais , Apoptose , Caspase 3/metabolismo , Caspase 7/metabolismo , Modelos Animais de Doenças , Iodatos , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Degeneração Retiniana/diagnóstico , Degeneração Retiniana/etiologia , Epitélio Pigmentado da Retina/patologia , Tomografia de Coerência Óptica
20.
Exp Eye Res ; 189: 107849, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31655042

RESUMO

7-Ketocholesterol (7KCh), an oxidized form of cholesterol, is present at a high level in drusen and has been believed to play a role in the pathogenesis of age-related macular degeneration (AMD). Therefore, we developed a rat model to study the direct impact of 7KCh on retina. We delivered 7KCh to the rat retina by intravitreal injection using hydroxypropyl-ß-cyclodextrin as a vehicle. We observed that 7KCh mainly deposited in the retinal pigment epithelial (RPE) cells and induced marked photoreceptor apoptosis. Transmission electron microscope examination demonstrated cytoplasmic vacuoles in RPE cells and the microvilli detached from the outer segment after 7KCh treatment. In vitro experiments also revealed that RPE cells could take up 7KCh in culture. Moreover, 7KCh up-regulated IL-1ßmRNA, TNF-αmRNA, IL-6 mRNA, and IL-1ß secretion of RPE. U0126, a MEK1/2 inhibitor, down regulated the expression of these inflammation factors. Our findings may help elucidate the potential role of 7KCh in the pathogenesis of AMD.


Assuntos
Regulação da Expressão Gênica , Inflamação/genética , Cetocolesteróis/genética , Degeneração Macular/genética , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Animais , Western Blotting , Células Cultivadas , Colesterol 7-alfa-Hidroxilase , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Inflamação/metabolismo , Inflamação/patologia , Cetocolesteróis/biossíntese , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Masculino , Microscopia Eletrônica de Transmissão , Fagocitose/fisiologia , RNA/genética , Ratos , Ratos Sprague-Dawley , Segmento Externo das Células Fotorreceptoras da Retina/patologia , Epitélio Pigmentado da Retina/ultraestrutura , Transdução de Sinais
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