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1.
J Vis Exp ; (168)2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33645569

RESUMO

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.


Assuntos
Dissecação , Cultura Primária de Células/métodos , Epitélio Pigmentado da Retina/citologia , Animais , Bioensaio , Diferenciação Celular , Polaridade Celular , Separação Celular , Impedância Elétrica , Células Epiteliais/citologia , Humanos , Camundongos Endogâmicos C57BL , Fagocitose , Fatores de Tempo
2.
J Vis Exp ; (168)2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33720134

RESUMO

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients >60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.


Assuntos
Separação Celular/métodos , Engenharia Genética , Terapia Genética , Mamíferos/metabolismo , Epitélio Pigmentado da Retina/citologia , Animais , Bovinos , Sobrevivência Celular , Células Cultivadas , Eletroporação , Proteínas do Olho/genética , Proteínas do Olho/uso terapêutico , Genes Reporter , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Camundongos , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/uso terapêutico , Estresse Oxidativo/genética , Ratos , Serpinas/genética , Serpinas/uso terapêutico , Suínos , Transfecção
3.
Int J Mol Sci ; 22(4)2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33579019

RESUMO

Progenitor cells derived from the retinal pigment epithelium (RPECs) have shown promise as therapeutic approaches to degenerative retinal disorders including diabetic retinopathy, age-related macular degeneration and Stargardt disease. However, the degeneration of Bruch's membrane (BM), the natural substrate for the RPE, has been identified as one of the major limitations for utilizing RPECs. This degeneration leads to decreased support, survival and integration of the transplanted RPECs. It has been proposed that the generation of organized structures of nanofibers, in an attempt to mimic the natural retinal extracellular matrix (ECM) and its unique characteristics, could be utilized to overcome these limitations. Furthermore, nanoparticles could be incorporated to provide a platform for improved drug delivery and sustained release of molecules over several months to years. In addition, the incorporation of tissue-specific genes and stem cells into the nanostructures increased the stability and enhanced transfection efficiency of gene/drug to the posterior segment of the eye. This review discusses available drug delivery systems and combination therapies together with challenges associated with each approach. As the last step, we discuss the application of nanofibrous scaffolds for the implantation of RPE progenitor cells with the aim to enhance cell adhesion and support a functionally polarized RPE monolayer.


Assuntos
Portadores de Fármacos/química , Nanofibras/química , Doenças Retinianas/terapia , Epitélio Pigmentado da Retina/transplante , Transplante de Células-Tronco/métodos , Tecidos Suporte/química , Animais , Lâmina Basilar da Corioide/química , Retinopatia Diabética/terapia , Sistemas de Liberação de Medicamentos/métodos , Humanos , Degeneração Macular/terapia , Epitélio Pigmentado da Retina/citologia , Doença de Stargardt/terapia , Células-Tronco/citologia
4.
Biochem Biophys Res Commun ; 545: 8-13, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33545636

RESUMO

Dysregulation of Wnt signaling is implicated in multiple ocular disorders. The roles of Wnt co-receptors LRP5 and LRP6 in Wnt signaling regulation remain elusive, as most retinal cells express both of the co-receptors. To address this question, LRP5 and LRP6 were individually knocked-out in a human retinal pigment epithelium cell line using the CRISPR-Cas9 technology. Wnt signaling activity induced by various Wnt ligands was measured using wild-type and the KO cell lines. The results identified three groups of Wnt ligands based on their co-receptor specificity: 1) activation of Wnt signaling only through LRP6, 2) through both LRP5 and LRP6 and 3) predominantly through LRP5. These results indicate that LRP5 and LRP6 have differential roles in Wnt signaling regulation.


Assuntos
Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Via de Sinalização Wnt , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Meios de Cultivo Condicionados , Receptores Frizzled/metabolismo , Técnicas de Inativação de Genes , Humanos , Ligantes , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/deficiência , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/deficiência , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Camundongos , Epitélio Pigmentado da Retina/citologia , Retinoides/metabolismo , Retinoides/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt3A/metabolismo
5.
J Vis Exp ; (168)2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33616098

RESUMO

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately understood, thus impeding the establishment of defined treatments. Cell-based additive gene therapies for the increased expression of a protective factor are considered as a promising option to medicate neurodegenerative diseases, such as age-related macular degeneration (AMD). We have developed a method for the stable expression of the gene encoding pigment epithelium-derived factor (PEDF), which is characterized as a neuroprotective and anti-angiogenic protein in the nervous system, into the genome of primary human pigment epithelial (PE) cells using the Sleeping Beauty (SB) transposon system. Primary PE cells were isolated from human donor eyes and maintained in culture. After reaching confluence, 1 x 104 cells were suspended in 11 µL of resuspension buffer and combined with 2 µL of a purified solution containing 30 ng of hyperactive SB (SB100X) transposase plasmid and 470 ng of PEDF transposon plasmid. Genetic modification was carried out with a capillary electroporation system using the following parameters: two pulses with a voltage of 1,100 V and a width of 20 ms. Transfected cells were transferred into culture plates containing medium supplemented with fetal bovine serum; antibiotics and antimycotics were added with the first medium exchange. Successful transfection was demonstrated in independently performed experiments. Quantitative polymerase chain reaction (qPCR) showed the increased expression of the PEDF transgene. PEDF secretion was significantly elevated and remained stable, as evaluated by immunoblotting, and quantified by enzyme-linked immunosorbent assay (ELISA). SB100X-mediated transfer allowed for a stable PEDF gene integration into the genome of PE cells and ensured the continuous secretion of PEDF, which is critical for the development of a cell-based gene addition therapy to treat AMD or other retinal degenerative diseases. Moreover, analysis of the integration profile of the PEDF transposon into human PE cells indicated an almost random genomic distribution.


Assuntos
Elementos de DNA Transponíveis , Eletroporação/métodos , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Transgenes , Transposases/metabolismo , Proteínas do Olho/metabolismo , Humanos , Fatores de Crescimento Neural/metabolismo , Serpinas/metabolismo , Transfecção , Transposases/genética
6.
Yakugaku Zasshi ; 141(1): 55-60, 2021.
Artigo em Japonês | MEDLINE | ID: mdl-33390448

RESUMO

The eye is a complex organ with highly specialized constituent tissues derived from different primordial cell lineages. The retina develops from neuroectoderm via the optic vesicle, the corneal epithelium is descended from surface ectoderm, while the iris and corneal stroma have a neural crest origin. Recent work with pluripotent stem cells (PSCs) in culture has revealed a previously under-appreciated level of intrinsic cellular self-organization, with a focus on the retina and retinal cells. We recently demonstrated the generation from human induced pluripotent stem cells (iPSCs) of a self-formed ectodermal autonomous multi-zone (SEAM) of ocular cells. The concentric SEAM mimics whole-eye development because cell location within different zones is indicative of ocular cell lineage, spanning the ocular surface ectoderm, lens, neuro-retina, and retinal pigment epithelium. Therefore, SEAM represents a promising resource for new research of ocular morphogenesis and development. Moreover, we successfully isolated corneal epithelial progenitor cells and fabricated corneal epithelial tissue from PSCs. This approach has translational potential for treating severe corneal epithelial disease by transplantation of PSC-derived corneal epithelial tissue. To evaluate the efficacy and safety of the corneal epithelial tissue, we have started a first-in-human clinical study for patients with corneal epithelial stem cell deficiency, which began last year.


Assuntos
Diferenciação Celular/fisiologia , Epitélio Anterior , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Medicina Regenerativa/métodos , Linhagem da Célula , Células Cultivadas , Doenças da Córnea/terapia , Epitélio Anterior/citologia , Epitélio Anterior/transplante , Humanos , Organoides , Epitélio Pigmentado da Retina/citologia
7.
Nat Commun ; 12(1): 662, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33510165

RESUMO

Dynamic assembly and disassembly of primary cilia controls embryonic development and tissue homeostasis. Dysregulation of ciliogenesis causes human developmental diseases termed ciliopathies. Cell-intrinsic regulatory mechanisms of cilia disassembly have been well-studied. The extracellular cues controlling cilia disassembly remain elusive, however. Here, we show that lysophosphatidic acid (LPA), a multifunctional bioactive phospholipid, acts as a physiological extracellular factor to initiate cilia disassembly and promote neurogenesis. Through systematic analysis of serum components, we identify a small molecular-LPA as the major driver of cilia disassembly. Genetic inactivation and pharmacological inhibition of LPA receptor 1 (LPAR1) abrogate cilia disassembly triggered by serum. The LPA-LPAR-G-protein pathway promotes the transcription and phosphorylation of cilia disassembly factors-Aurora A, through activating the transcription coactivators YAP/TAZ and calcium/CaM pathway, respectively. Deletion of Lpar1 in mice causes abnormally elongated cilia and decreased proliferation in neural progenitor cells, thereby resulting in defective neurogenesis. Collectively, our findings establish LPA as a physiological initiator of cilia disassembly and suggest targeting the metabolism of LPA and the LPA pathway as potential therapies for diseases with dysfunctional ciliogenesis.


Assuntos
Cílios/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Neurogênese/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Transdução de Sinais , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cílios/genética , Cílios/metabolismo , Células HEK293 , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Lisofosfolipídeos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Ligação Proteica , Interferência de RNA , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo
8.
Phytomedicine ; 80: 153375, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33096452

RESUMO

BACKGROUND: Dry age-related macular degeneration (dAMD) leads to serious burden of visual impairment and there is no definitive treatment. Previous studies have showed that naringenin (NAR) significantly increased electroretinography (ERG) c-wave in sodium iodate (NaIO3)-treated rats and viability of NaIO3-treated ARPE-19 cells. But the underlying mechanism is still unknown. PURPOSE: We tested the hypothesis that anti-oxidation mediated by Sirtuin 1 (SIRT1) was important to the protective effect of NAR on dAMD. STUDY DESIGN/METHODS: NaIO3-induced mice retinopathy and ARPE-19 cells injury models were established. In vivo, the protective effect of NAR eye drops on retina was evaluated by flash ERG (FERG) recording and histopathological examination. In vitro, viability of ARPE-19 cells, and the levels of lactic dehydrogenase (LDH), reactive oxygen species (ROS) and carbonyl protein were detected. Protein expression of SIRT1 was analyzed by immunochemical staining, immunofluorescence and western blotting. RESULTS: NAR eye drops improved retinal function and morphology and normalized the protein expression of SIRT1 in mice exposed to NaIO3. NAR promoted the survival of ARPE-19 cells in a concentration-dependent manner. NAR up-regulated SIRT1 protein expression, and decreased levels of ROS and carbonyl protein. Moreover, EX527, a selective inhibitor of SIRT1, abolished the effects of NAR on the cell viability and ROS. In addition, SRT1720, a selective agonist of SIRT1, improved the viability of cells and suppressed the production of ROS. CONCLUSION: Our findings indicate that SIRT1-mediated anti-oxidation contributes to the protective effect of NAR eye drops on dAMD.


Assuntos
Flavanonas/farmacologia , Substâncias Protetoras/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Sirtuína 1/metabolismo , Animais , Carbazóis/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Iodatos/toxicidade , L-Lactato Desidrogenase/metabolismo , Masculino , Camundongos , Soluções Oftálmicas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/tratamento farmacológico , Epitélio Pigmentado da Retina/citologia , Regulação para Cima/efeitos dos fármacos
9.
J Vis Exp ; (165)2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33311434

RESUMO

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2-9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day. The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.


Assuntos
Técnicas de Cultura de Órgãos/métodos , Epitélio Pigmentado da Retina/crescimento & desenvolvimento , Animais , Técnicas de Cultura de Células , Meios de Cultura Livres de Soro , Camundongos , Epitélio Pigmentado da Retina/citologia
10.
Life Sci ; 259: 118391, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32891610

RESUMO

AIMS: Dyslipidemia-associated diabetic retinopathy is featured by macular edema and retinal angiogenesis. This study investigated the in vitro lipotoxicity of free fatty acids and their modulatory roles in regulation of autophagy and angiogenic factor production in cultured human retinal pigment epithelium (RPE) ARPE-19 cells. MAIN METHODS: ARPE-19 cells were exposed to monounsaturated oleic acid (OA), saturated palmitic acid (PA), or both. Cell viability, cell cycle distribution, migration, and autophagy of the treated cells were monitored. Angiogenic factor production was determined by RT-qPCR and ELISA. KEY FINDINGS: OA, but not PA, at doses higher than 500 µM significantly induced cytostasis and lipotoxicity in ARPE-19 cells. OA exposure not only markedly enhanced autophagy flux, but also enhanced cell migration, while PA suppressed motility of RPE cells. Meanwhile, OA stimulated de novo synthesis of angiogenic factors including VEGF and bFGF in ARPE-19 cells. Mechanistically, OA treatment stimulated not only AMPK/mTOR/p70S6K signaling, but also induced hyperphosphorylation of MAPK pathway mediators, including ERK, JNK and p38 MAPK, as well as NF-κB activation. Kinase inhibition assays showed that blockade of PI3K/Akt, MAPK and NF-κB pathways prevented the OA-upregulated VEGF transcription and its peptide release. Comparatively, only NF-κB inhibition significantly suppressed bFGF peptide release from ARPE-19 cells. SIGNIFICANCE: Out findings support the OA-exhibited cytostasis, autophagy modulation and angiogenic factor production in RPE cells. This study sheds light on the interrelationship between metabolic disorder and retinopathy and provides molecular strategies for preventing and treating choroidal neovascularization in diabetic retinopathy.


Assuntos
Indutores da Angiogênese/metabolismo , Autofagia , Ácido Oleico/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Western Blotting , Movimento Celular , Proliferação de Células , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Sistema de Sinalização das MAP Quinases , Ácido Palmítico/metabolismo , Epitélio Pigmentado da Retina/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
11.
Med Sci (Paris) ; 36(6-7): 594-599, 2020.
Artigo em Francês | MEDLINE | ID: mdl-32614310

RESUMO

The neuroretina is a functional unit of the central nervous system that converts a light signal into a nerve impulse. Of neuroectodermal origin, derived from the diencephalon, the neuroretina is a layered tissue composed of six types of neuronal cells (two types of photoreceptors: cones and rods, horizontal, bipolar, amacrine and ganglion cells) and three types of glial cells (Müller glial cells, astrocytes and microglial cells). The neuroretina lays on the retinal pigmentary epithelium, that together form the retina. The existence of the internal and external blood-retinal barriers and intra-retinal junctions reflects the fineness of regulation of the retinal exchanges with the circulation and within the retina itself. The central zone of the human retina, which is highly specialized for visual acuity, has anatomical specificities. Recent imaging methods make it possible now to enrich our knowledge of the anatomical and functional characteristics of the retina, which are still imperfectly described.


Assuntos
Retina/anatomia & histologia , Animais , Corioide/irrigação sanguínea , Corioide/citologia , Corioide/fisiologia , Humanos , Neuroglia/citologia , Neuroglia/fisiologia , Retina/citologia , Retina/fisiologia , Retina/ultraestrutura , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Epitélio Pigmentado da Retina/irrigação sanguínea , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Vasos Retinianos/citologia , Vasos Retinianos/fisiologia
12.
Sci Rep ; 10(1): 7656, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32376945

RESUMO

Transplantation of autologous human induced pluripotent stem cell-derived retinal pigment epithelial (hiPSC-RPE) sheets is a promising therapy for age-related macular degeneration (AMD). As melanin content is a representative feature of healthy RPE, we used polarization-sensitive optical coherence tomography (PS-OCT) to estimate the relative melanin content of RPE in diseased and non-diseased area, and in human iPSC-RPE sheets in vitro and in vivo by evaluating the randomness of polarization (entropy). Two aged Japanese women, one with neovascular AMD that underwent transplantation of an autologous hiPSC-RPE cell sheet and another with binocular dry AMD, were selected for this study. Entropy value was minimal in cells containing no melanin, whereas that of human RPE and hiPSC-RPE sheets was high. En face entropy of the cultured hiPSC-RPE sheet was compared with its grey-scale photo and its values were found to be inversely correlated with the extent of absence of pigmentation in vitro. En face entropy maps were compared to colour fundus photographs, fundus autofluorescence images, and fluorescein angiography images from patients. Entropy values of intact and defective RPEs and of iPSC-RPE transplant areas were determined in vivo using PS-OCT B-scan images. PS-OCT was found to be applicable in the estimation of relative melanin content of cultured and transplanted RPEs in regenerative medicine.


Assuntos
Biomarcadores , Células-Tronco Pluripotentes Induzidas/citologia , Melaninas/metabolismo , Epitélio Pigmentado da Retina/diagnóstico por imagem , Epitélio Pigmentado da Retina/metabolismo , Tomografia de Coerência Óptica , Idoso , Idoso de 80 Anos ou mais , Técnicas de Cultura de Células , Diferenciação Celular , Feminino , Angiofluoresceinografia , Células HEK293 , Humanos , Degeneração Macular/diagnóstico por imagem , Degeneração Macular/etiologia , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Masculino , Epitélio Pigmentado da Retina/citologia , Tomografia de Coerência Óptica/métodos
13.
PLoS One ; 15(5): e0233057, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32396545

RESUMO

Poor survival of human pluripotent stem cells (hPSCs) following freezing, thawing, or passaging hinders the maintenance and differentiation of stem cells. Rho-associated kinases (ROCKs) play a crucial role in hPSC survival. To date, a typical ROCK inhibitor, Y-27632, has been the primary agent used in hPSC research. Here, we report that another ROCK inhibitor, fasudil, can be used as an alternative and is cheaper than Y-27632. It increased hPSC growth following thawing and passaging, like Y-27632, and did not affect pluripotency, differentiation ability, and chromosome integrity. Furthermore, fasudil promoted retinal pigment epithelium (RPE) differentiation and the survival of neural crest cells (NCCs) during differentiation. It was also useful for single-cell passaging of hPSCs and during aggregation. These findings suggest that fasudil can replace Y-27632 for use in stem research.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Amidas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Crista Neural/citologia , Crista Neural/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Pesquisa com Células-Tronco
14.
Nat Commun ; 11(1): 2196, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32366837

RESUMO

Transition fibers (TFs) regulate cilia gating and make the primary cilium a distinct functional entity. However, molecular insights into the biogenesis of a functional cilia gate remain elusive. In a forward genetic screen in Caenorhabditis elegans, we uncover that TALP-3, a homolog of the Joubert syndrome protein TALPID3, is a TF-associated component. Genetic analysis reveals that TALP-3 coordinates with ANKR-26, the homolog of ANKRD26, to orchestrate proper cilia gating. Mechanistically, TALP-3 and ANKR-26 form a complex with key gating component DYF-19, the homolog of FBF1. Co-depletion of TALP-3 and ANKR-26 specifically impairs the recruitment of DYF-19 to TFs. Interestingly, in mammalian cells, TALPID3 and ANKRD26 also play a conserved role in coordinating the recruitment of FBF1 to TFs. We thus report a conserved protein module that specifically regulates the functional component of the ciliary gate and suggest a correlation between defective gating and ciliopathy pathogenesis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Cílios/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Animais Geneticamente Modificados , Corpos Basais/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Células Cultivadas , Centríolos/metabolismo , Cílios/genética , Células HEK293 , Humanos , Microscopia Confocal , Mutação , Interferência de RNA , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo
15.
Invest Ophthalmol Vis Sci ; 61(4): 32, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32334435

RESUMO

Purpose: Oxidative stress in retinal pigment epithelial (RPE) cells is associated with age-related macular degeneration (AMD). Resveratrol exerts a range of protective biologic effects, but its mechanism(s) are not well understood. The aim of this study was to investigate how resveratrol could affect biologic pathways in oxidatively stressed RPE cells. Methods: Cultured human RPE cells were treated with hydroquinone (HQ) in the presence or absence of resveratrol. Cell viability was determined with WST-1 reagent and trypan blue exclusion. Mitochondrial function was measured with the XFe24 Extracellular Flux Analyzer. Expression of heme oxygenase-1 (HO-1) and glutamate cysteine ligase catalytic subunit was evaluated by qPCR. Endoplasmic reticulum stress protein expression was measured by Western blot. Potential reactions between HQ and resveratrol were investigated using high-performance liquid chromatography mass spectrometry with resveratrol and additional oxidants for comparison. Results: RPE cells treated with the combination of resveratrol and HQ had significantly increased cell viability and improved mitochondrial function when compared with HQ-treated cells alone. Resveratrol in combination with HQ significantly upregulated HO-1 mRNA expression above that of HQ-treated cells alone. Resveratrol in combination with HQ upregulated C/EBP homologous protein and spliced X-box binding protein 1. Additionally, new compounds were formed from resveratrol and HQ coincubation. Conclusions: Resveratrol can ameliorate HQ-induced toxicity in RPE cells through improved mitochondrial bioenergetics, upregulated antioxidant genes, stimulated unfolded protein response, and direct oxidant interaction. This study provides insight into pathways through which resveratrol can protect RPE cells from oxidative damage, a factor thought to contribute to AMD pathogenesis.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Heme Oxigenase-1/genética , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Resveratrol/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Western Blotting/métodos , Células Cultivadas , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidroquinonas/farmacologia , Oxirredução , Reação em Cadeia da Polimerase em Tempo Real/métodos , Epitélio Pigmentado da Retina/citologia
16.
PLoS One ; 15(4): e0231212, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32275682

RESUMO

Two major proteolytic systems, the proteasome and the autophagy pathway, are key components of the proteostasis network. The immunoproteasome, a proteasome subtype, and autophagy are upregulated under stress conditions, forming a coordinated unit designed to minimize the effect of cell stress. We investigated how genetic ablation of the LMP2 immunoproteasome subunit affects autophagy in retinal pigment epithelium (RPE) from WT and LMP2 knockout mice. We monitored autophagy regulation by measuring LC3, phosphorylation of AKT (S473), and phosphorylation of S6, a downstream readout of AKT (mTOR) pathway activation. We also evaluated transcription factor EB (TFEB) nuclear translocation, a transcription factor that controls expression of autophagy and lysosome genes. WT and LMP2 KO cells were monitored after treatment with EBSS to stimulate autophagy, insulin to stimulate AKT, or an AKT inhibitor (trehalose or MK-2206). Under basal conditions, we observed hyper-phosphorylation of AKT and S6, as well as lower nuclear-TFEB content in LMP2 KO RPE compared with WT. AKT inhibitors MK-2206 and trehalose significantly inhibited AKT phosphorylation and stimulated nuclear translocation of TFEB. Starvation and AKT inhibition upregulated autophagy, albeit to a lesser extent in LMP2 KO RPE. These data support the idea that AKT hyper-activation is an underlying cause of defective autophagy regulation in LMP2 KO RPE, revealing a unique link between two proteolytic systems and a previously unknown function in autophagy regulation by the immunoproteasome.


Assuntos
Autofagia , Complexo de Endopeptidases do Proteassoma/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Retina/citologia , Animais , Autofagia/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Células Cultivadas , Cisteína Endopeptidases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Insulina/farmacologia , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Epitélio Pigmentado da Retina/citologia , Transdução de Sinais/efeitos dos fármacos
17.
Biochem Biophys Res Commun ; 525(3): 675-680, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32139118

RESUMO

Glucocorticoids require the glucocorticoid receptor (GR), a type of ligand-dependent nuclear receptor to transmit their downstream effects. Upon glucocorticoid binding, GR associates with glucocorticoid response elements (GREs) and recruits other transcriptional coregulators to activate or repress target gene transcription. Many SET-domain family proteins have been demonstrated to contribute to GR-mediated transcriptional activity. However, whether histone H3K4-specific methyltransferase plays a cell-type-specific role in GR transcriptional regulation remains poorly understood. In this report, we examined MLL2 (KMT2D), a histone-lysine methyltransferase that catalyzes histone H3 lysine 4 methylation (H3K4me). Furthermore, we demonstrated that MLL2 specifically regulates the transcription of some GR target genes (e.g., ENACα and FLJ20371) in ARPE-19 cells, but has no effect in A549 cells. Mechanistically, co-immunoprecipitation assays revealed that MLL2 is associated with GR in a ligand-independent manner in APRE-19 cells. Moreover, chromatin immunoprecipitation analyses demonstrated that MLL2 could co-occupy glucocorticoid response elements (GREs) of GR target genes along with GR following Dex stimulation. Finally, the FAIRE-qPCR results illustrated that MLL2 is pivotal in establishing chromatin structure accessibility at the GREs of ARPE-19 specific genes in the presence of Dex. Taken together, our study determined that MLL2 regulates GR-mediated transcription in a cell-type-specific manner, and we provide a molecular mechanism to explain the specific role of MLL2 in regulating GR target gene expression in ARPE-19 cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Glucocorticoides/metabolismo , Epitélio Pigmentado da Retina/citologia , Transcrição Genética , Sítios de Ligação , Linhagem Celular , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Dexametasona/farmacologia , Regulação da Expressão Gênica , Humanos
18.
J Cell Biol ; 219(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32211891

RESUMO

Distal appendages (DAs) of the mother centriole are essential for the initial steps of ciliogenesis in G1/G0 phase of the cell cycle. DAs are released from centrosomes in mitosis by an undefined mechanism. Here, we show that specific DAs lose their centrosomal localization at the G2/M transition in a manner that relies upon Nek2 kinase activity to ensure low DA levels at mitotic centrosomes. Overexpression of active Nek2A, but not kinase-dead Nek2A, prematurely displaced DAs from the interphase centrosomes of immortalized retina pigment epithelial (RPE1) cells. This dramatic impact was also observed in mammary epithelial cells with constitutively high levels of Nek2. Conversely, Nek2 knockout led to incomplete dissociation of DAs and cilia in mitosis. As a consequence, we observed the presence of a cilia remnant that promoted the asymmetric inheritance of ciliary signaling components and supported cilium reassembly after cell division. Together, our data establish Nek2 as an important kinase that regulates DAs before mitosis.


Assuntos
Centríolos/enzimologia , Cílios/enzimologia , Células Epiteliais/enzimologia , Mitose , Quinases Relacionadas a NIMA/metabolismo , Epitélio Pigmentado da Retina/enzimologia , Animais , Sítios de Ligação , Linhagem Celular , Centríolos/genética , Cílios/genética , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular , Células-Tronco Hematopoéticas/enzimologia , Humanos , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/enzimologia , Camundongos , Proteínas dos Microtúbulos/genética , Proteínas dos Microtúbulos/metabolismo , Quinases Relacionadas a NIMA/genética , Ligação Proteica , Epitélio Pigmentado da Retina/citologia , Transdução de Sinais , Fatores de Tempo
19.
Exp Eye Res ; 193: 107985, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32092287

RESUMO

Strong communication and interaction between the retinal pigment epithelium (RPE) and the photoreceptor (PR) cells is essential for vision. RPE cells are essential for supporting and maintaining PR cells by transporting nutrients, waste products and ions, and phagocytosing photoreceptor outer segments (POS). POS phagocytosis follows a circadian pattern, taking place in the morning in human, mice and other organisms. However, it remains unknown whether other RPE processes follow a daily rhythm. To study the daily rhythm of RPE cells, we isolated murine RPE cells at six different time points during a 24 h period, after which RNA was isolated and sequenced. Murine RPE flatmounts were isolated at four different time points to study daily rhythm in protein abundance and localisation. EnrichR pathway analysis resulted in 13 significantly-enriched KEGG pathways (p < 0.01) of which seven showed a large number of overlapping genes. Several genes were involved in intracellular trafficking, possibly playing a role in nutrient transport, POS phagocytosis or membrane protein trafficking, with different expression patterns during the day-night cycle. Other genes were involved in actin cytoskeleton building, remodelling and crosslinking and showed a high expression in the morning, suggesting actin cytoskeleton remodelling at this time point. Finally, tight junction proteins Cldn2 and Cldn4 showed a difference in RNA and protein expression and tight junction localisation over time. Our study suggests that several important processes in the RPE follow a day-night rhythm, including intracellular trafficking, and processes involving the actin cytoskeleton and tight junctions. The differential protein localisation of Cldn2 in the RPE during the day-night cycle suggest that Cldn2 may facilitate paracellular water and sodium transport during the day.


Assuntos
Ritmo Circadiano/fisiologia , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Proteínas de Junções Íntimas/genética , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Epitélio Pigmentado da Retina/citologia , Proteínas de Junções Íntimas/biossíntese
20.
Sci Rep ; 10(1): 2758, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32066800

RESUMO

PDCD4, the protein encoded by the tumor suppressor gene PDCD4 (programmed cell death 4) has been implicated in the control of cellular transcription and translation by modulating the activity of specific transcription factors and suppressing the translation of mRNAs with structured 5'-UTRs. Most studies of human PDCD4 have employed tumor cell lines, possibly resulting in a biased picture of its role in normal cells. Here, we have studied the function of PDCD4 in a telomerase-immortalized human epithelial cell line. We show for the first time that PDCD4 is required for the G1/S-transition, demonstrating its crucial role in the cell cycle. Inhibition of p53-dependent activation of p21WAF1/CIP1 overrides the requirement for PDCD4 for the G1/S-transition, suggesting that PDCD4 counteracts basal p53 activity to prevent activation of the G1/S checkpoint by p53. Transcriptome and ribosome profiling data show that silencing of PDCD4 changes the expression levels and translation of many mRNAs, providing an unbiased view of the cellular processes that are affected by PDCD4 in an epithelial cell line. Our data identify PDCD4 as a key regulator of cell cycle- and DNA-related functions that are inhibited when it is silenced, suggesting that decreased expression of PDCD4 might contribute to tumor development by compromising genomic integrity.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Células Epiteliais/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Biossíntese de Proteínas , Proteínas de Ligação a RNA/genética , Telomerase/genética , Transcriptoma , Regiões 5' não Traduzidas , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Transformada , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células Epiteliais/citologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Transdução de Sinais , Telomerase/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
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