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1.
PLoS One ; 15(2): e0226311, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32053618

RESUMO

It is not currently possible to reliably visualise and track immune cells in the human central nervous system or eye. Previous work demonstrated that indocyanine green (ICG) dye could label immune cells and be imaged after a delay during disease in the mouse retina. We report a pilot study investigating if ICG can similarly label immune cells within the human retina. Twelve adult participants receiving ICG angiography as part of routine standard of care were recruited. Baseline retinal images were obtained prior to ICG administration then repeated over a period ranging from 2 hours to 9 days. Matched peripheral blood samples were obtained to examine systemic immune cell labelling and activation from ICG by flow cytometry with human macrophage cultures as positive controls. Differences between the delayed near infrared ICG imaging and 488 nm autofluorescence was observed across pathologies, likely arising from the retinal pigment epithelium (RPE). Only one subject demonstrated ICG signal on peripheral blood myeloid cells and only three distinct cell-sized signals appeared over time within the retina of three participants. No significant increase in immune cell activation markers were detected after ICG administration. ICG accumulated in the endosomes of macrophage cultures and was detectable above a minimum concentration, suggesting cell labelling is possible. ICG can label RPE and may be used as an additional biomarker for RPE health across a range of retinal disorders. Standard clinical doses of intravenous ICG do not lead to robust immune cell labelling in human blood or retina and further optimisation in dose and route are required.


Assuntos
Corantes/administração & dosagem , Verde de Indocianina/administração & dosagem , Leucócitos Mononucleares/química , Macrófagos/química , Epitélio Pigmentado da Retina/diagnóstico por imagem , Adulto , Idoso , Corantes/química , Endossomos/química , Estudos de Viabilidade , Feminino , Citometria de Fluxo , Angiofluoresceinografia , Humanos , Verde de Indocianina/química , Injeções Intravenosas , Macrófagos/citologia , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Estudos Prospectivos , Epitélio Pigmentado da Retina/citologia , Coloração e Rotulagem/métodos , Adulto Jovem
2.
Invest Ophthalmol Vis Sci ; 61(2): 9, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32049341

RESUMO

Purpose: Variant B precursor cysteine protease inhibitor cystatin C, a known recessive risk factor for developing exudative age-related macular degeneration (AMD), presents altered intracellular trafficking and reduced secretion from retinal pigment epithelial (RPE) cells. Because cystatin C inhibits multiple extracellular matrix (ECM)-degrading cathepsins, this study evaluated the role of this mutation in inducing ECM-related functional changes in RPE cellular behavior. Methods: Induced pluripotent stem cells gene-edited bi-allelically by CRISPR/Cas9 to express the AMD-linked cystatin C variant were differentiated to RPE cells and assayed for their ability to degrade fluorescently labeled ECM proteins. Cellular migration and adhesion on multiple ECM proteins, differences in transepithelial resistance and polarized protein secretion were tested. Vessel formation induced by gene edited cells-conditioned media was quantified using primary human dermal microvascular epithelial cells. Results: Variant B cystatin C-expressing induced pluripotent stem cells-derived RPE cells displayed a significantly higher rate of laminin and fibronectin degradation 3 days after seeding on fluorescently labeled ECM (P < 0.05). Migration on matrigel, collagen IV and fibronectin was significantly faster for edited cells compared with wild-type (WT) cells. Both edited and WT cells displayed polarized secretion of cystatin C, but transepithelial resistance was lower in gene-edited cells after 6 weeks culture, with significantly lower expression of tight junction protein claudin-3. Media conditioned by gene-edited cells stimulated formation of significantly longer microvascular tubes (P < 0.05) compared with WT-conditioned media. Conclusions: Reduced levels of cystatin C lead to changes in the RPE ability to degrade, adhere, and migrate supporting increased invasiveness and angiogenesis relevant for AMD pathology.


Assuntos
Cistatina C/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Degeneração Macular/patologia , Epitélio Pigmentado da Retina/citologia , Movimento Celular/fisiologia , Células Cultivadas , Cistatina C/genética , Cistatina C/metabolismo , Fibronectinas/metabolismo , Edição de Genes , Humanos , Laminina/metabolismo , Mutação Puntual/genética
3.
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
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.
Gene ; 724: 144157, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31629820

RESUMO

Cellular microRNAs are known to modulate the life-cycle of different viruses. Surprisingly, very little data exists on AAV-induced changes to the cellular microRNAome in general and in hepatic and retinal cells, in particular. We reasoned that inducible microRNA in response to recombinant AAV infection may regulate immediate and long-lived cellular responses necessary for the cell's own survival as well as its ability to control several aspects of viral life-cycle. To study this, we performed a global small RNA sequencing analysis in Adeno-associated virus (AAV) serotypes 2 and 3 infected hepatic and retinal cell models. This screen identified multiple differentially expressed microRNAs, in AAV infected Huh-7 and ARPE-19 cells. Among these, one microRNA (miR-4488) was found to be significantly down regulated (-2.24 fold for AAV2 and -3.32 fold for ARPE-19) in AAV infected cells. An enrichment and pathway analysis of miR-4488 predicted its possible effects on gene targets involved in multiple biological processes including cell-cycle regulation, endoplasmic reticulum stress response and lipid-signalling pathways. Moreover, validation studies in miR-4488 mimic or sponge transfected cells revealed modulation of these target pathways in a cell-specific manner. Further studies demonstrated that overexpression of miR-4488, modestly increased gene expression (126-128%) from AAV2 and AAV3 vectors in Huh-7 cells whereas miR-4488 inhibition in ARPE-19 cells had a similar increase (142-158%) on AAV2 or AAV3 transduction. Our results highlight that recombinant AAV mediated microRNA expression is cell-type and serotype-specific and can target specific host cellular biological pathways.


Assuntos
Dependovirus/genética , MicroRNAs/genética , Infecções por Parvoviridae/genética , Epitélio Pigmentado da Retina/virologia , Transdução Genética/métodos , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/genética , Perfilação da Expressão Gênica , Humanos , Metabolismo dos Lipídeos/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Parvovirinae/genética , Reprodutibilidade dos Testes , Epitélio Pigmentado da Retina/citologia , Transgenes
6.
Adv Exp Med Biol ; 1185: 227-231, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884616

RESUMO

Pre-mRNA splicing is a critical step in RNA processing in all eukaryotic cells. It consists of introns removal and requires the assembly of a large RNA-protein complex called the spliceosome. This complex of small nuclear ribonucleoproteins is associated with accessory proteins from the pre-mRNA processing factor (PRPF) family. Mutations in different splicing factor-encoding genes were identified in retinitis pigmentosa (RP) patients. A surprising feature of these ubiquitous factors is that the outcome of their alteration is restricted to the retina. Because of their high metabolic demand, most studies focused on photoreceptors dysfunction and associated degeneration. However, cells from the retinal pigment epithelium (RPE) are also crucial to maintaining retinal homeostasis and photoreceptor function. Moreover, mutations in RPE-specific genes are associated with some RP cases. Indeed, we identified major RPE defects in Prpf31-mutant mice: circadian rhythms of both photoreceptor outer segments (POS) phagocytosis and retinal adhesion were attenuated or lost, leading to ultrastructural anomalies and vacuoles. Taken together, our published and ongoing data suggest that (1) similar molecular events take place in human and mouse cells and (2) these functional defects generate various stress processes.


Assuntos
Células Epiteliais/patologia , Proteínas do Olho/genética , Retinite Pigmentosa/genética , Animais , Ritmo Circadiano , Células Epiteliais/ultraestrutura , Humanos , Camundongos , Fagocitose , Células Fotorreceptoras de Vertebrados/patologia , Fatores de Processamento de RNA/genética , Epitélio Pigmentado da Retina/citologia , Retinite Pigmentosa/patologia
7.
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
8.
Adv Exp Med Biol ; 1185: 389-393, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884643

RESUMO

The retinal pigment epithelium (RPE) performs several functions that are crucial for normal retinal function and vision, including the daily phagocytosis of photoreceptor outer segment (POS) membranes. Defects in the motility and degradation of POS phagosomes may be associated with some inherited and age-related retinal degenerations. Given the apical to basal translocation of phagosomes during maturation and degradation, studies of the underlying mechanisms require analyses of the dynamics in 3-D. In this chapter, we report a method for investigating the 3-D motility of POS phagosomes and lysosomes, utilizing high-speed, spinning disk confocal microscopy of live RPE flatmounts.


Assuntos
Lisossomos/fisiologia , Fagossomos/fisiologia , Segmento Externo das Células Fotorreceptoras da Retina/fisiologia , Epitélio Pigmentado da Retina/diagnóstico por imagem , Humanos , Microscopia Confocal , Fagocitose , Epitélio Pigmentado da Retina/citologia
9.
Adv Exp Med Biol ; 1185: 419-423, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884648

RESUMO

Bestrophinopathies are a group of clinically distinct inherited retinal dystrophies that lead to the gradual loss of vision in and around the macular area. There are no treatments for patients suffering from bestrophinopathies, and no measures can be taken to prevent visual deterioration in those who have inherited disease-causing mutations. Bestrophinopathies are caused by mutations in the Bestrophin1 gene (BEST1), a protein found exclusively in the retinal pigment epithelial (RPE) cells of the eye. Mutations in BEST1 affect the function of the RPE leading to the death of overlying retinal cells and subsequent vision loss. The pathogenic mechanisms arising from BEST1 mutations are still not fully understood, and it is not clear how mutations in BEST1 lead to diseases with distinct clinical features. This chapter discusses BEST1, the use of model systems to investigate the effects of mutations and the potential to investigate individual bestrophinopathies using induced pluripotent stem cells.


Assuntos
Bestrofinas/genética , Células-Tronco Pluripotentes Induzidas , Doenças Retinianas/genética , Canais de Cloreto , Proteínas do Olho , Humanos , Mutação , Epitélio Pigmentado da Retina/citologia
10.
Adv Exp Med Biol ; 1185: 525-530, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884665

RESUMO

Calcium is one of the most important second messengers in cells and thus involved in a variety of physiological processes. In retinal pigment epithelium (RPE), Ca2+ and its ATP-dependent signaling pathways play important roles in the retina maintenance functions. Changes in intracellular Ca2+ concentration can be measured from living cells by Ca2+ imaging. Combining these measurements with quantitative analysis of Ca2+ response properties enables studies of signaling pathways affecting RPE functions. However, robust tools for response analysis from large cell populations are lacking. We developed MATLAB-based analysis tools for single cell level Ca2+ response data recorded from large fields of intact RPE monolayers. The analysis revealed significant heterogeneity in ATP-induced Ca2+ responses inside cell populations regarding magnitude and response kinetics. Further analysis including response grouping and parameter correlations allowed us to characterize the populations at the level of single cells.


Assuntos
Trifosfato de Adenosina/metabolismo , Sinalização do Cálcio , Epitélio Pigmentado da Retina/citologia , Células Cultivadas , Humanos
11.
Adv Exp Med Biol ; 1185: 557-561, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884670

RESUMO

Patient-derived human-induced pluripotent stem cells (iPSCs) have been critical in advancing our understanding of the underlying mechanisms of numerous retinal disorders. Many of these retinal disorders have no effective treatment and result in severe visual impairment and even blindness. Among the retinal degenerative diseases modeled by iPSCs are age-related macular degeneration (AMD), glaucoma, Leber congenital amaurosis (LCA), retinitis pigmentosa (RP), and autosomal dominant retinitis pigmentosa (adRP). In addition to studying retinal disease ontogenesis and pathology, hiPSCs have clinical and pharmacological applications, such as developing drug screening and gene therapy approaches and new cell-based clinical treatments. Recent studies have primarily focused on three retinal cell fates - retinal pigmented epithelium cells (RPE), retinal ganglion cells (RGCs), and photoreceptor cells - and have demonstrated that hiPSCs have great potential for increasing our knowledge of and developing treatments for retinal degenerative disorders.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Retina/citologia , Degeneração Retiniana/terapia , Humanos , Células-Tronco Pluripotentes Induzidas/transplante , Células Fotorreceptoras/citologia , Células Ganglionares da Retina/citologia , Epitélio Pigmentado da Retina/citologia
12.
Adv Exp Med Biol ; 1185: 563-567, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884671

RESUMO

The replacement of retinal cells, or the support of surviving retinal neurons, in a degenerated retina presents a significant challenge in the fields of ophthalmology and regenerative medicine. Stem cell-based therapies are being explored as an approach for treating retinal dystrophies, such as retinitis pigmentosa (RP), Stargardt's disease, and age-related macular degeneration (AMD). This review provides an update on the recent progress made toward the restoration of vision lost to degenerative disease using stem cell-based transplantation strategies and the challenges that need to be overcome. Both retinal pigmented epithelium (RPE) and photoreceptor replacement therapies are discussed.


Assuntos
Células-Tronco Pluripotentes/citologia , Doenças Retinianas/terapia , Transplante de Células-Tronco , Humanos , Células Fotorreceptoras , Retina , Epitélio Pigmentado da Retina/citologia
13.
Adv Exp Med Biol ; 1185: 569-574, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884672

RESUMO

The goal of this study was to quantitatively assess retinal thickness using spectral domain optical coherence tomography (SD-OCT) after subretinal implantation of human embryonic stem cell-derived retinal pigment epithelium in a porcine model. The implant is called CPCB-RPE1 for the California Project to Cure Blindness-Retinal Pigment Epithelium 1. Data were derived from previous experiments on 14 minipigs that received either subretinal implantation of CPCB-RPE1 (n = 11) or subretinal bleb formation alone (sham; n = 3) using previously described methods and procedures (Brant Fernandes et al. Ophthalmic Surg Lasers Imaging Retina 47:342-51, 2016; Martynova et al. (2016) Koss et al. Graefes Arch Clin Exp Ophthalmol 254:1553-65, 2016; Hu et al. Ophthalmic Res 48:186-91, 2016; Martynova et al. ARVO Abstract 2016. SD-OCT retinal thickness (RT) and sublayer thickness over the implant were compared with topographically similar preimplantation regions as described previously Martynova et al. ARVO Abstract 2016. Imaging results were compared to postmortem histology using hematoxylin-eosin staining. RT overlying the CPCB-RPE1 postimplantation was not significantly different from preimplantation (308 ± 72 µm vs 292 ± 41 µm; p = 0.44). RT was not significantly different before and after implantation in any retinal sublayer at 1 month. Histology demonstrated grossly normal retinal anatomy as well as photoreceptor interdigitation with RPE.


Assuntos
Células-Tronco Embrionárias Humanas/transplante , Retina/diagnóstico por imagem , Epitélio Pigmentado da Retina/citologia , Tomografia de Coerência Óptica , Animais , California , Humanos , Suínos
14.
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
15.
Nihon Yakurigaku Zasshi ; 154(4): 192-196, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31597898

RESUMO

Primary cilia are non-motile cilia consisting of a centriole-derived basal body and a microtubule-based axoneme. In recent years, the structure and function of primary cilia have been attracting attention due to the relation with the onset of ciliary disease. Scanning ion conductance microscopy (SICM) is a probe microscopy used to measure the topography and functions of living cells at nanoscale. Furthermore, the labelling procedure is not necessary for SICM measurement compare to fluorescence imaging. We compared the structures of primary cilia of human retinal pigment epithelial cell line (RPE-1 cells) and Madin-Darby canine kidney cell line (MDCK cells) at nanoscale by using SICM. In addition, high resolution SICM images have also succeeded in visualizing ciliary pockets that difficult to be fluorescently labeled.


Assuntos
Cílios/fisiologia , Microscopia de Varredura por Sonda , Animais , Cães , Humanos , Íons , Células Madin Darby de Rim Canino , Epitélio Pigmentado da Retina/citologia
16.
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
17.
Adv Exp Med Biol ; 1186: 1-31, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31654384

RESUMO

Pluripotent stem cell technology, including human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs), has provided a suitable platform to investigate molecular and pathological alterations in an individual cell type using patient's own cells. Importantly, hiPSCs/hESCs are amenable to genome editing providing unique access to isogenic controls. Specifically, the ability to introduce disease-causing mutations in control (unaffected) and conversely correct disease-causing mutations in patient-derived hiPSCs has provided a powerful approach to clearly link the disease phenotype with a specific gene mutation. In fact, utilizing hiPSC/hESC and CRISPR technology has provided significant insight into the pathomechanism of several diseases. With regard to the eye, the use of hiPSCs/hESCs to study human retinal diseases is especially relevant to retinal pigment epithelium (RPE)-based disorders. This is because several studies have now consistently shown that hiPSC-RPE in culture displays key physical, gene expression and functional attributes of human RPE in vivo. In this book chapter, we will discuss the current utility, limitations, and plausible future approaches of pluripotent stem cell technology for the study of retinal degenerative diseases. Of note, although we will broadly summarize the significant advances made in modeling and studying several retinal diseases utilizing hiPSCs/hESCs, our specific focus will be on the utility of patient-derived hiPSCs for (1) establishment of human cell models and (2) molecular and pharmacological studies on patient-derived cell models of retinal degenerative diseases where RPE cellular defects play a major pathogenic role in disease development and progression.


Assuntos
Células-Tronco Pluripotentes , Degeneração Retiniana , Epitélio Pigmentado da Retina , Diferenciação Celular , Humanos , Células-Tronco Pluripotentes Induzidas , Retina/patologia , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/patologia
18.
Adv Exp Med Biol ; 1186: 33-53, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31654385

RESUMO

The advent of stem cell technology, including the technology to induce pluripotency in somatic cells, and direct differentiation of stem cells into specific somatic cell types, has created an exciting new field of scientific research. Much of the work with pluripotent stem (PS) cells has been focused on the exploration and exploitation of their potential as cells/tissue replacement therapies for personalized medicine. However, PS and stem cell-derived somatic cells are also proving to be valuable tools to study disease pathology and tissue-specific responses to injury. High-throughput drug screening assays using tissue-specific injury models have the potential to identify specific and effective treatments that will promote wound healing. Retinal pigment epithelium (RPE) derived from induced pluripotent stem cells (iPS-RPE) are well characterized cells that exhibit the phenotype and functions of in vivo RPE. In addition to their role as a source of cells to replace damaged or diseased RPE, iPS-RPE provide a robust platform for in vitro drug screening to identify novel therapeutics to promote healing and repair of ocular tissues after injury. Proliferative vitreoretinopathy (PVR) is an abnormal wound healing process that occurs after retinal tears or detachments. In this chapter, the role of iPS-RPE in the development of an in vitro model of PVR is described. Comprehensive analyses of the iPS-RPE response to injury suggests that these cells provide a physiologically relevant tool to investigate the cellular mechanisms of the three phases of PVR pathology: migration, proliferation, and contraction. This in vitro model will provide valuable information regarding cellular wound healing responses specific to RPE and enable the identification of effective therapeutics.


Assuntos
Células-Tronco Pluripotentes Induzidas , Epitélio Pigmentado da Retina , Vitreorretinopatia Proliferativa , Diferenciação Celular , Células Cultivadas , Humanos , Técnicas In Vitro , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/patologia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/patologia , Vitreorretinopatia Proliferativa/patologia
19.
Mater Sci Eng C Mater Biol Appl ; 105: 110131, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31546376

RESUMO

Silk fibroin membrane displays potential for ocular tissue reconstruction as demonstrated by its ability to support a functioning retinal pigment epithelium (RPE) in vitro. Nevertheless, translation of these findings to the clinic will require the use of membranes that can be readily handled and implanted into diseased retinas, with minimal impact on the surrounding healthy tissue. To this end, we optimized the physical properties of fibroin membranes to enable surgical handling during implantation into the retina, without compromising biocompatibility or permeability. Our central hypothesis is that optimal strength and permeability can be achieved by combining the porogenic properties of poly(ethylene glycol) (PEG) with the crosslinking properties of horseradish peroxidase (HRP). Our study reveals that PEG used in conjunction with HRP enables the production of fibroin membranes with superior handling properties to conventional fibroin membranes. More specifically, the modified membranes could be more easily implanted into the retinas of rats and displayed good evidence of biocompatibility. Moreover, the modified membranes retained the ability to support construction of functional RPE derived from pluripotent stem cells. These findings pave the way for preclinical studies of RPE-implantation using the optimized fibroin membranes.


Assuntos
Fibroínas/química , Membranas Artificiais , Próteses Visuais , Animais , Bombyx , Células-Tronco Embrionárias Humanas/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Permeabilidade , Fagocitose , Ratos , Epitélio Pigmentado da Retina/citologia , Soluções , Espectroscopia de Infravermelho com Transformada de Fourier , Resistência à Tração
20.
Artif Cells Nanomed Biotechnol ; 47(1): 3758-3764, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31556307

RESUMO

Diabetic retinopathy (DR) is one of the most common diabetic complications and remains the leading cause of vision loss among adults. C1q/TNF-related protein 3 (CTRP3) is a member of CTRP family that has been found to be involved in the progression of diabetes mellitus and diabetic complications. However, the role of CTRP3 in DR has not been fully understood. In the present study, the results showed that CTRP3 expression was significantly decreased in DR patients compared with controls. In vitro investigations proved that overexpression of CTRP3 improved cell viability of ARPE-19 cells in response to high glucose (HG) stimulation. CTRP3 also attenuated HG-induced oxidative stress in ARPE-19 cells with decreased levels of reactive oxygen species (ROS) and malondialdehyde (MDA), and increased superoxide dismutase (SOD) activity. Apoptotic rate was significantly decreased in CTRP3 overexpressing ARPE-19 cells. Besides, bcl-2 expression was increased, while bax expression was decreased by CTRP3 overexpression. Moreover, overexpression of CTRP3 enhanced the activation of nuclear factor erythroid 2-related factor 2 (Nrf2)/hemeoxygenase-1 (HO-1) pathway in HG-stimulated ARPE-19 cells, and Nrf2 knockdown reversed CTRP3-mediated oxidative stress and apoptosis. These findings suggested that CTRP3 attenuated HG-stimulated oxidative stress and apoptosis in ARPE-19 cells, which were mediated by activation of Nrf2/HO-1 pathway.


Assuntos
Apoptose/efeitos dos fármacos , Glucose/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Epitélio Pigmentado da Retina/citologia , Fatores de Necrose Tumoral/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Heme Oxigenase-1/metabolismo , Humanos , Fator 2 Relacionado a NF-E2/deficiência , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo
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