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1.
Commun Biol ; 4(1): 850, 2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34239035

RESUMO

The retinal pigmented epithelium (RPE) is a monolayer of multifunctional cells located at the back of the eye. High membrane turnover and polarization, including formation of actin-based apical microvilli, are essential for RPE function and retinal health. Herein, we demonstrate an important role for ßA3/A1-crystallin in RPE. ßA3/A1-crystallin deficiency leads to clathrin-mediated epidermal growth factor receptor (EGFR) endocytosis abnormalities and actin network disruption at the apical side that result in RPE polarity disruption and degeneration. We found that ßA3/A1-crystallin binds to phosphatidylinositol transfer protein (PITPß) and that ßA3/A1-crystallin deficiency diminishes phosphatidylinositol 4,5-biphosphate (PI(4,5)P2), thus probably decreasing ezrin phosphorylation, EGFR activation, internalization, and degradation. We propose that ßA3/A1-crystallin acquired its RPE function before evolving as a structural element in the lens, and that in the RPE, it modulates the PI(4,5)P2 pool through PITPß/PLC signaling axis, coordinates EGFR activation, regulates ezrin phosphorylation and ultimately the cell polarity.


Assuntos
Polaridade Celular/fisiologia , Endocitose , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Cadeia A de beta-Cristalina/metabolismo , Animais , Polaridade Celular/genética , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/ultraestrutura , Humanos , Camundongos Knockout , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosforilação , Ligação Proteica , Epitélio Pigmentado da Retina/citologia , Cadeia A de beta-Cristalina/genética
2.
Nat Commun ; 11(1): 1195, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32139672

RESUMO

Here, we report that the functionality of vascular progenitors (VP) generated from normal and disease-primed conventional human induced pluripotent stem cells (hiPSC) can be significantly improved by reversion to a tankyrase inhibitor-regulated human naïve epiblast-like pluripotent state. Naïve diabetic vascular progenitors (N-DVP) differentiated from patient-specific naïve diabetic hiPSC (N-DhiPSC) possessed higher vascular functionality, maintained greater genomic stability, harbored decreased lineage-primed gene expression, and were more efficient in migrating to and re-vascularizing the deep neural layers of the ischemic retina than isogenic diabetic vascular progenitors (DVP). These findings suggest that reprogramming to a stable naïve human pluripotent stem cell state may effectively erase dysfunctional epigenetic donor cell memory or disease-associated aberrations in patient-specific hiPSC. More broadly, tankyrase inhibitor-regulated naïve hiPSC (N-hiPSC) represent a class of human stem cells with high epigenetic plasticity, improved multi-lineage functionality, and potentially high impact for regenerative medicine.


Assuntos
Vasos Sanguíneos/patologia , Diabetes Mellitus/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Isquemia/terapia , Retina/patologia , Células-Tronco/patologia , Tanquirases/antagonistas & inibidores , Adulto , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Linhagem da Célula/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Dano ao DNA , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Código das Histonas , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Isquemia/patologia , Camundongos , Organoides/efeitos dos fármacos , Organoides/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Regiões Promotoras Genéticas/genética , Células-Tronco/efeitos dos fármacos , Células-Tronco/ultraestrutura , Tanquirases/metabolismo , Teratoma/patologia , Transcrição Genética/efeitos dos fármacos
3.
Autophagy ; 16(6): 1130-1142, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31462148

RESUMO

Persistent fetal vasculature (PFV) is a human disease that results from failure of the fetal vasculature to regress normally. The regulatory mechanisms responsible for fetal vascular regression remain obscure, as does the underlying cause of regression failure. However, there are a few animal models that mimic the clinical manifestations of human PFV, which can be used to study different aspects of the disease. One such model is the Nuc1 rat model that arose from a spontaneous mutation in the Cryba1 (crystallin, beta 1) gene and exhibits complete failure of the hyaloid vasculature to regress. Our studies with the Nuc1 rat indicate that macroautophagy/autophagy, a process in eukaryotic cells for degrading dysfunctional components to ensure cellular homeostasis, is severely impaired in Nuc1 ocular astrocytes. Further, we show that CRYBA1 interacts with EGFR (epidermal growth factor receptor) and that loss of this interaction in Nuc1 astrocytes increases EGFR levels. Moreover, our data also show a reduction in EGFR degradation in Nuc1 astrocytes compared to control cells that leads to over-activation of the mechanistic target of rapamycin kinase complex 1 (MTORC1) pathway. The impaired EGFR-MTORC1-autophagy signaling in Nuc1 astrocytes triggers abnormal proliferation and migration. The abnormally migrating astrocytes ensheath the hyaloid artery, contributing to the pathogenesis of PFV in Nuc1, by adversely affecting the vascular remodeling processes essential to regression of the fetal vasculature. Herein, we demonstrate in vivo that gefitinib (EGFR inhibitor) can rescue the PFV phenotype in Nuc1 and may serve as a novel therapy for PFV disease by modulating the EGFR-MTORC1-autophagy pathway. ABBREVIATIONS: ACTB: actin, beta; CCND3: cyclin 3; CDK6: cyclin-dependent kinase 6; CHQ: chloroquine; COL4A1: collagen, type IV, alpha 1; CRYBA1: crystallin, beta A1; DAPI: 4'6-diamino-2-phenylindole; EGFR: epidermal growth factor receptor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary growth factor; KDR: kinase insert domain protein receptor; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MKI67: antigen identified by monoclonal antibody Ki 67; MTORC1: mechanistic target of rapamycin kinase complex 1; PARP: poly (ADP-ribose) polymerase family; PCNA: proliferating cell nuclear antigen; PFV: persistent fetal vasculature; PHPV: persistent hyperplastic primary vitreous; RPE: retinal pigmented epithelium; RPS6: ribosomal protein S6; RPS6KB1: ribosomal protein S6 kinase, polypeptide 1; SQSTM1/p62: sequestome 1; TUBB: tubulin, beta; VCL: vinculin; VEGFA: vascular endothelial growth factor A; WT: wild type.


Assuntos
Astrócitos/metabolismo , Autofagia/genética , Receptores ErbB/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Vítreo Primário Hiperplásico Persistente/metabolismo , Cadeia A de beta-Cristalina/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Modelos Animais de Doenças , Receptores ErbB/antagonistas & inibidores , Olho/metabolismo , Gefitinibe/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/genética , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Microscopia Imunoeletrônica , Morfolinas/farmacologia , Vítreo Primário Hiperplásico Persistente/genética , Vítreo Primário Hiperplásico Persistente/patologia , Vítreo Primário Hiperplásico Persistente/terapia , Ratos , Transdução de Sinais/genética , Sirolimo/farmacologia , Cadeia A de beta-Cristalina/genética
4.
Exp Eye Res ; 181: 252-262, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30807744

RESUMO

The choriocapillaris is the source of nutrients and oxygen for photoreceptors, which consume more oxygen per gram of tissue than any other cell in the body. The purpose of this study was to evaluate and compare the ultrastructure of the choriocapillaris and its transport systems in patients with and without age-related macular degeneration (AMD). Ultrastructural changes were also evaluated in subjects that were homozygous for polymorphisms in high risk CFH alleles (Pure 1) only or homozygous only for high risk ARMS2/HTRA1 (Pure 10) alleles. Tissue samples were obtained from the macular region of forty male (n = 24) and female (n = 16) donor eyes and prepared for ultrastructural studies with transmission electron microscopy (TEM). The average age of the aged donors was 74 ±â€¯7.2 (n = 30) and the young donors 31.7 ±â€¯11.25 (n = 10). There was no significant difference in average ages between the adult groups. TEM images of the capillaries in the choriocapillaris (CC) were taken at 4,000X and 25,000X and used to measure the area of endothelial cell somas, the number of fenestrations, and area of caveolae within the endothelial cells per length of Bruchs membrane (BrMb). The Student t-test and Wilcoxon sum rank test were used to determine significant differences. There was no significant difference between young subjects and aged controls in any of the morphological criteria assessed. There was a significant decrease in the number of fenestrations/mm of BrMb in atrophic areas of GA eyes (p = 0.007) when compared with aged control eyes. A significant increase was found in the caveolae area as a percent of the endothelial cell soma of capillaries from GA subjects as compared with the controls (p = 0.03). Loss of capillary segments in choriocapillaris was also evident, especially in areas of geographic atrophy and CNV. In eyes from patients with sequence variations, the capillary endothelial cells often appeared degenerative and exhibited atypical fenestrations and pericytes covering the blood vessels. Subjects that were homozygous for polymorphisms in high risk CFH alleles only had more fenestrations/mm of BrMb than subjects that were homozygous only for high risk ARMS2/HTRA1 alleles (p = 0.04), while the latter had greater caveolae area/endothelial cell area than the former (p = 0.007). This study demonstrated an attenuation of CC and a significant decline in the two major transport systems in CC endothelial cells in AMD. This may contribute to drusen deposition, nutrient transport, and vision loss in AMD subjects.


Assuntos
Corioide/ultraestrutura , Oxigênio/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Degeneração Macular Exsudativa/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Corioide/metabolismo , Feminino , Humanos , Transporte de Íons , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Epitélio Pigmentado da Retina/ultraestrutura , Adulto Jovem
5.
Sci Rep ; 7(1): 766, 2017 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-28396597

RESUMO

The retinal degenerative diseases, which together constitute a leading cause of hereditary blindness worldwide, are largely untreatable. Development of reliable methods to culture complex retinal tissues from human pluripotent stem cells (hPSCs) could offer a means to study human retinal development, provide a platform to investigate the mechanisms of retinal degeneration and screen for neuroprotective compounds, and provide the basis for cell-based therapeutic strategies. In this study, we describe an in vitro method by which hPSCs can be differentiated into 3D retinas with at least some important features reminiscent of a mature retina, including exuberant outgrowth of outer segment-like structures and synaptic ribbons, photoreceptor neurotransmitter expression, and membrane conductances and synaptic vesicle release properties consistent with possible photoreceptor synaptic function. The advanced outer segment-like structures reported here support the notion that 3D retina cups could serve as a model for studying mature photoreceptor development and allow for more robust modeling of retinal degenerative disease in vitro.


Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes/citologia , Retina/citologia , Retina/metabolismo , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Biomarcadores , Técnicas de Cultura de Células , Células Cultivadas , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/ultraestrutura , Expressão Gênica , Humanos , Hipóxia , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Fatores de Tempo
6.
Invest Ophthalmol Vis Sci ; 58(3): 1352-1367, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28249091

RESUMO

Purpose: Müller cells create the external limiting membrane (ELM) by forming junctions with photoreceptor cells. This study evaluated the relationship between focal photoreceptors and RPE loss in geographic atrophy (GA) and Müller cell extension into the subretinal space. Methods: Human donor eyes with no retinal disease or geographic atrophy (GA) were fixed and the eye cups imaged. The retinal posterior pole was stained for glial fibrillary acidic protein (GFAP; astrocytes and activated Müller cells) and vimentin (Müller cells) while the submacular choroids were labeled with Ulex Europaeus Agglutinin lectin (blood vessels). Choroids and retinas were imaged using a Zeiss 710 confocal microscope. Additional eyes were cryopreserved or processed for transmission electron microscopy (TEM) to better visualize the Müller cells. Results: Vimentin staining of aged control retinas (n = 4) revealed a panretinal cobblestone-like ELM. While this pattern was also observed in the GA retinas (n = 7), each also had a distinct area in which vimentin+ and vimentin+/GFAP+ processes created a subretinal membrane. Subretinal glial membranes closely matched areas of RPE atrophy in the gross photos. Choroidal vascular loss was also evident in these atrophic areas. Smaller glial projections were noted, which correlated with drusen in gross photos. The presence of glia in the subretinal space was confirmed by TEM and cross cross-section immunohistochemistry. Conclusions: In eyes with GA, subretinal Müller cell membranes present in areas of RPE atrophy may be a Müller cell attempt to replace the ELM. These membranes could interfere with treatments such as stem cell therapy.


Assuntos
Células Ependimogliais/ultraestrutura , Atrofia Geográfica/patologia , Retina/ultraestrutura , Idoso , Idoso de 80 Anos ou mais , Astrócitos/metabolismo , Astrócitos/ultraestrutura , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Células Ependimogliais/metabolismo , Feminino , Atrofia Geográfica/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Retina/metabolismo , Vimentina/metabolismo
7.
Aging Cell ; 16(2): 349-359, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28083894

RESUMO

The dry (nonneovascular) form of age-related macular degeneration (AMD), a leading cause of blindness in the elderly, has few, if any, treatment options at present. It is characterized by early accumulation of cellular waste products in the retinal pigmented epithelium (RPE); rejuvenating impaired lysosome function in RPE is a well-justified target for treatment. It is now clear that amino acids and vacuolar-type H+ -ATPase (V-ATPase) regulate the mechanistic target of rapamycin, complex 1 (mTORC1) signaling in lysosomes. Here, we provide evidence for the first time that the amino acid transporter SLC36A4/proton-dependent amino acid transporter (PAT4) regulates the amino acid pool in the lysosomes of RPE. In Cryba1 (gene encoding ßA3/A1-crystallin) KO (knockout) mice, where PAT4 and amino acid levels are increased in the RPE, the transcription factors EB (TFEB) and E3 (TFE3) are retained in the cytoplasm, even after 24 h of fasting. Consequently, genes in the coordinated lysosomal expression and regulation (CLEAR) network are not activated, and lysosomal function remains low. As these mice age, expression of RPE65 and lecithin retinol acyltransferase (LRAT), two vital visual cycle proteins, decreases in the RPE. A defective visual cycle would possibly slow down the regeneration of new photoreceptor outer segments (POS). Further, photoreceptor degeneration also becomes obvious during aging, reminiscent of human dry AMD disease. Electron microscopy shows basal laminar deposits in Bruch's membrane, a hallmark of development of AMD. For dry AMD patients, targeting PAT4/V-ATPase in the lysosomes of RPE cells may be an effective means of preventing or delaying disease progression.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Células Epiteliais/metabolismo , Complexos Multiproteicos/metabolismo , Epitélio Pigmentado da Retina/citologia , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Envelhecimento/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Cristalinas/metabolismo , Citosol/metabolismo , Células Epiteliais/ultraestrutura , Redes Reguladoras de Genes , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos Knockout , Fosforilação , Ligação Proteica , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/ultraestrutura , Vias Visuais/metabolismo , Cadeia A de beta-Cristalina
8.
Invest Ophthalmol Vis Sci ; 57(4): 2213-24, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-27116549

RESUMO

PURPOSE: The choriocapillaris (CC), the capillary network of the choroid, is positioned adjacent to Bruch's membrane (BM) and the RPE. The aim of this study was to clarify the mechanism(s) for transport of serum albumen from CC lumen to RPE. METHODS: Alexa647 conjugated to BSA (BSA-A647) or PBS was administrated via the femoral vein to young and aged wild-type (WT; C57BL/6J) mice and Caveolin-1 knockout mice (Cav1(-/-)). Mice were perfused with PBS and killed at 30 minutes, 1 hour, and 4 hours after injection. Eyecups were cryopreserved, and cryosections were analyzed on a Zeiss 710 confocal microscope. Bovine serum albumin conjugated to gold nanoparticles (BSA-GNP) was administrated through the left common carotid artery. Mice were perfused with PBS and killed at 30 minutes after injection. Eyecups were embedded after fixation, and 70-nm-thick sections were analyzed on a Hitachi H7600 transmission electron microscope. RESULTS: In eyes of WT young mice, BSA-A647 was transported to the RPE at 30 minutes and diffused to the photoreceptor layer by 1 hour. In contrast, most BSA-A647 was found in the CC in Cav1(-/-) eyes. The majority of BSA-GNP found in the CC of young WT mice was on the luminal side in caveolae at 30 minutes after injection. In aged WT mice, BSA-GNPs were found in defective tight junctions between endothelial cells and appeared trapped at the diaphragm of fenestrations. CONCLUSIONS: Normally, CC carefully regulates transport system of BSA from lumen to BM by caveolae-mediated transcytosis; however, endothelium cells of aged control WT mice have leaky tight junctions and lacked regulated BSA transport.


Assuntos
Lâmina Basilar da Corioide/fisiologia , Capilares/fisiologia , Corioide/irrigação sanguínea , Epitélio Pigmentado Ocular/fisiologia , Albumina Sérica/metabolismo , Animais , Lâmina Basilar da Corioide/metabolismo , Lâmina Basilar da Corioide/ultraestrutura , Capilares/ultraestrutura , Caveolina 1/fisiologia , Corioide/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Imagem Óptica/métodos , Epitélio Pigmentado Ocular/metabolismo , Epitélio Pigmentado Ocular/ultraestrutura
9.
Exp Eye Res ; 144: 46-53, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26321509

RESUMO

The retinal pigmented epithelium (RPE) is critically important to retinal homeostasis, in part due to its very active processes of phagocytosis and autophagy. Both of these processes depend upon the normal functioning of lysosomes, organelles which must fuse with (auto)phagosomes to deliver the hydrolases that effect degradation of cargo. It has become clear that signaling through mTOR complex 1 (mTORC1), is very important in the regulation of lysosomal function. This signaling pathway is becoming a target for therapeutic intervention in diseases, including age-related macular degeneration (AMD), where lysosomal function is defective. In addition, our laboratory has been studying animal models in which the gene (Cryba1) for ßA3/A1-crystallin is deficient. These animals exhibit impaired lysosomal clearance in the RPE and pathological signs that are similar to some of those seen in AMD patients. The data demonstrate that ßA3/A1-crystallin localizes to lysosomes in the RPE and that it is a binding partner of V-ATPase, the proton pump that acidifies the lysosomal lumen. This suggests that ßA3/A1-crystallin may also be a potential target for therapeutic intervention in AMD. In this review, we focus on effector molecules that impact the lysosomal-autophagic pathway in RPE cells.


Assuntos
Autofagia/fisiologia , Lisossomos/fisiologia , Epitélio Pigmentado da Retina/metabolismo , Animais , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/fisiologia , Biogênese de Organelas , Serina-Treonina Quinases TOR/fisiologia
10.
Aging Cell ; 13(6): 1091-4, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25257511

RESUMO

Although chronic inflammation is believed to contribute to the pathology of age-related macular degeneration (AMD), knowledge regarding the events that elicit the change from para-inflammation to chronic inflammation in the pathogenesis of AMD is lacking. We propose here that lipocalin-2 (LCN2), a mammalian innate immunity protein that is trafficked to the lysosomes, may contribute to this process. It accumulates significantly with age in retinal pigment epithelial (RPE) cells of Cryba1 conditional knockout (cKO) mice, but not in control mice. We have recently shown that these mice, which lack ßA3/A1-crystallin specifically in RPE, have defective lysosomal clearance. The age-related increase in LCN2 in the cKO mice is accompanied by increases in chemokine (C-C motif) ligand 2 (CCL2), reactive gliosis, and immune cell infiltration. LCN2 may contribute to induction of a chronic inflammatory response in this mouse model with AMD-like pathology.


Assuntos
Proteínas de Fase Aguda/metabolismo , Cristalinas/metabolismo , Lipocalinas/metabolismo , Degeneração Macular/metabolismo , Proteínas Oncogênicas/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Fatores Etários , Animais , Doença Crônica , Cristalinas/genética , Modelos Animais de Doenças , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Lipocalina-2 , Degeneração Macular/genética , Degeneração Macular/patologia , Camundongos , Epitélio Pigmentado da Retina/patologia , Cadeia A de beta-Cristalina
11.
PLoS Genet ; 10(5): e1004360, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24875170

RESUMO

During organogenesis, PAX6 is required for establishment of various progenitor subtypes within the central nervous system, eye and pancreas. PAX6 expression is maintained in a variety of cell types within each organ, although its role in each lineage and how it acquires cell-specific activity remain elusive. Herein, we aimed to determine the roles and the hierarchical organization of the PAX6-dependent gene regulatory network during the differentiation of the retinal pigmented epithelium (RPE). Somatic mutagenesis of Pax6 in the differentiating RPE revealed that PAX6 functions in a feed-forward regulatory loop with MITF during onset of melanogenesis. PAX6 both controls the expression of an RPE isoform of Mitf and synergizes with MITF to activate expression of genes involved in pigment biogenesis. This study exemplifies how one kernel gene pivotal in organ formation accomplishes a lineage-specific role during terminal differentiation of a single lineage.


Assuntos
Diferenciação Celular/genética , Proteínas do Olho/biossíntese , Proteínas de Homeodomínio/biossíntese , Fator de Transcrição Associado à Microftalmia/genética , Organogênese/genética , Fatores de Transcrição Box Pareados/biossíntese , Proteínas Repressoras/biossíntese , Animais , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Camundongos , Fator de Transcrição Associado à Microftalmia/biossíntese , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Pigmentação/genética , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Epitélio Pigmentado da Retina/crescimento & desenvolvimento , Epitélio Pigmentado da Retina/metabolismo
12.
Autophagy ; 10(3): 480-96, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24468901

RESUMO

In phagocytic cells, including the retinal pigment epithelium (RPE), acidic compartments of the endolysosomal system are regulators of both phagocytosis and autophagy, thereby helping to maintain cellular homeostasis. The acidification of the endolysosomal system is modulated by a proton pump, the V-ATPase, but the mechanisms that direct the activity of the V-ATPase remain elusive. We found that in RPE cells, CRYBA1/ßA3/A1-crystallin, a lens protein also expressed in RPE, is localized to lysosomes, where it regulates endolysosomal acidification by modulating the V-ATPase, thereby controlling both phagocytosis and autophagy. We demonstrated that CRYBA1 coimmunoprecipitates with the ATP6V0A1/V0-ATPase a1 subunit. Interestingly, in mice when Cryba1 (the gene encoding both the ßA3- and ßA1-crystallin forms) is knocked out specifically in RPE, V-ATPase activity is decreased and lysosomal pH is elevated, while cathepsin D (CTSD) activity is decreased. Fundus photographs of these Cryba1 conditional knockout (cKO) mice showed scattered lesions by 4 months of age that increased in older mice, with accumulation of lipid-droplets as determined by immunohistochemistry. Transmission electron microscopy (TEM) of cryba1 cKO mice revealed vacuole-like structures with partially degraded cellular organelles, undigested photoreceptor outer segments and accumulation of autophagosomes. Further, following autophagy induction both in vivo and in vitro, phospho-AKT and phospho-RPTOR/Raptor decrease, while pMTOR increases in RPE cells, inhibiting autophagy and AKT-MTORC1 signaling. Impaired lysosomal clearance in the RPE of the cryba1 cKO mice also resulted in abnormalities in retinal function that increased with age, as demonstrated by electroretinography. Our findings suggest that loss of CRYBA1 causes lysosomal dysregulation leading to the impairment of both autophagy and phagocytosis.


Assuntos
Autofagia/fisiologia , Cristalinas/metabolismo , Lisossomos/metabolismo , Complexos Multiproteicos/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Autofagia/genética , Cristalinas/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , Fagocitose/fisiologia , Fagossomos/metabolismo , Ratos , Epitélio Pigmentado da Retina/citologia , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/genética
13.
Circulation ; 129(3): 359-72, 2014 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-24163065

RESUMO

BACKGROUND: The generation of vascular progenitors (VPs) from human induced pluripotent stem cells (hiPSCs) has great potential for treating vascular disorders such as ischemic retinopathies. However, long-term in vivo engraftment of hiPSC-derived VPs into the retina has not yet been reported. This goal may be limited by the low differentiation yield, greater senescence, and poor proliferation of hiPSC-derived vascular cells. To evaluate the potential of hiPSCs for treating ischemic retinopathies, we generated VPs from a repertoire of viral-integrated and nonintegrated fibroblast and cord blood (CB)-derived hiPSC lines and tested their capacity for homing and engrafting into murine retina in an ischemia-reperfusion model. METHODS AND RESULTS: VPs from human embryonic stem cells and hiPSCs were generated with an optimized vascular differentiation system. Fluorescence-activated cell sorting purification of human embryoid body cells differentially expressing endothelial/pericytic markers identified a CD31(+)CD146(+) VP population with high vascular potency. Episomal CB-induced pluripotent stem cells (iPSCs) generated these VPs with higher efficiencies than fibroblast-iPSC. Moreover, in contrast to fibroblast-iPSC-VPs, CB-iPSC-VPs maintained expression signatures more comparable to human embryonic stem cell VPs, expressed higher levels of immature vascular markers, demonstrated less culture senescence and sensitivity to DNA damage, and possessed fewer transmitted reprogramming errors. Luciferase transgene-marked VPs from human embryonic stem cells, CB-iPSCs, and fibroblast-iPSCs were injected systemically or directly into the vitreous of retinal ischemia-reperfusion-injured adult nonobese diabetic-severe combined immunodeficient mice. Only human embryonic stem cell- and CB-iPSC-derived VPs reliably homed and engrafted into injured retinal capillaries, with incorporation into damaged vessels for up to 45 days. CONCLUSIONS: VPs generated from CB-iPSCs possessed augmented capacity to home, integrate into, and repair damaged retinal vasculature.


Assuntos
Células-Tronco Embrionárias/citologia , Sangue Fetal/citologia , Células-Tronco Pluripotentes/citologia , Traumatismo por Reperfusão/terapia , Doenças Retinianas/terapia , Transplante de Células-Tronco/métodos , Animais , Capilares/citologia , Senescência Celular , Dano ao DNA , Modelos Animais de Doenças , Fibroblastos/citologia , Sobrevivência de Enxerto , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Regeneração , Traumatismo por Reperfusão/patologia , Doenças Retinianas/patologia , Transcriptoma
14.
Invest Ophthalmol Vis Sci ; 54(3): 1887-97, 2013 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-23422828

RESUMO

PURPOSE: We compared the cellular phenotypes and studied the role of autophagy in the pathogenesis of Fuchs endothelial corneal dystrophy (FECD) using two α2 collagen VIII (Col8a2) knock-in mouse models and human FECD tissues. METHODS: In vivo corneal endothelial cell (CEC) counts and morphology were analyzed by clinical confocal microscopy. Ultrastructural analysis of CECs was performed by transmission electron microscopy. Real-time PCR and Western blotting were performed using total RNA, and protein extracted from mouse CECs and human CECs obtained from FECD and autopsy patients. RESULTS: Both Col8a2 mouse models exhibited hallmarks of FECD; however, the Col8a2(L450W/L450W) mice exhibited a milder phenotype compared to the Col8a2(Q455K/Q455K) mice. Both models exhibited upregulation of the unfolded protein response (UPR) as evidenced by dilated rough endoplasmic reticulum (RER), and upregulation of UPR-associated genes and proteins. Real-time PCR of Col8a2(L450W/L450W) and Col8a2(Q455K/Q455K) CECs at 40 weeks revealed a 2.1-fold (P < 0.05) and a 5.2-fold (P < 0.01) upregulation of the autophagy marker Dram1, respectively. Real-time PCR of human FECD endothelium revealed a 10.4-fold upregulation of DRAM1 (P < 0.0001) compared to autopsy controls. CONCLUSIONS: The Col8a2(L450W/L450W) and Col8a2(Q455K/Q455K) mouse models of FECD showed distinct endothelial cell phenotypes. Dram1 was associated with activation of the UPR and increased autophagy. Overexpression of this gene in mouse and human FECD endothelial cells suggested a role for altered autophagy in this disease.


Assuntos
Autofagia/fisiologia , Colágeno Tipo VIII/genética , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/patologia , Animais , Autofagia/genética , Western Blotting , Colágeno Tipo VIII/fisiologia , Modelos Animais de Doenças , Células Endoteliais/patologia , Endotélio Corneano/metabolismo , Técnicas de Introdução de Genes , Genótipo , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real
15.
Invest Ophthalmol Vis Sci ; 53(13): 7912-27, 2012 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-23092923

RESUMO

PURPOSE: The mode of development of the human hyaloid vascular system (HVS) remains unclear. Early studies suggested that these blood vessels formed by vasculogenesis, while the current concept seems to favor angiogenesis as the mode of development. We examined embryonic and fetal human HVS using a variety of techniques to gain new insights into formation of this vasculature. METHODS: Embryonic and fetal human eyes from 5.5 to 12 weeks gestation (WG) were prepared for immunohistochemical analysis or for light and electron microscopy. Immunolabeling of sections with a panel of antibodies directed at growth factors, transcription factors, and hematopoietic stem cell markers was employed. RESULTS: Light microscopic examination revealed free blood islands (BI) in the embryonic vitreous cavity (5.5-7 WG). Giemsa stain revealed that BI were aggregates of mesenchymal cells and primitive nucleated erythroblasts. Free cells were also observed. Immunolabeling demonstrated that BI were composed of mesenchymal cells that expressed hemangioblast markers (CD31, CD34, C-kit, CXCR4, Runx1, and VEGFR2), erythroblasts that expressed embryonic hemoglobin (Hb-ε), and cells that expressed both. Few cells were proliferating as determined by lack of Ki67 antigen. As development progressed (12 WG), blood vessels became more mature structurally with pericyte investment and basement membrane formation. Concomitantly, Hb-ε and CXCR4 expression was down-regulated and von Willebrand factor expression was increased with the formation of Weibel-Palade bodies. CONCLUSIONS: Our results support the view that the human HVS, like the choriocapillaris, develops by hemo-vasculogenesis, the process by which vasculogenesis, erythropoiesis, and hematopoiesis occur simultaneously from common precursors, hemangioblasts.


Assuntos
Cristalino/irrigação sanguínea , Neovascularização Fisiológica/fisiologia , Artéria Oftálmica/embriologia , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Endotélio Vascular/metabolismo , Eritroblastos/metabolismo , Eritroblastos/ultraestrutura , Hemoglobina Fetal , Técnica Indireta de Fluorescência para Anticorpo , Idade Gestacional , Humanos , Técnicas Imunoenzimáticas , Cristalino/embriologia , Mesoderma/metabolismo , Mesoderma/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores CXCR4/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Corpo Vítreo/irrigação sanguínea , Corpo Vítreo/embriologia
16.
Hum Mol Genet ; 21(2): 384-93, 2012 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-22002996

RESUMO

Fuchs endothelial corneal dystrophy (FECD) is a leading indication for corneal transplantation. FECD is characterized by progressive alterations in endothelial cell morphology, excrescences (guttae) and thickening of the endothelial basement membrane and cell death. Ultimately, these changes lead to corneal edema and vision loss. Due to the lack of vision loss in early disease stages and the decades long disease course, early pathophysiology in FECD is virtually unknown as studies of pathologic tissues have been limited to end-stage tissues obtained at transplant. The first genetic defect shown to cause FECD was a point mutation causing a glutamine to lysine substitution at amino acid position 455 (Q455K) in the alpha 2 collagen 8 gene (COL8A2) which results in an early onset form of the disease. Homozygous mutant knock-in mice with this mutation (Col8a2(Q455K/Q455K)) show features strikingly similar to human disease, including progressive alterations in endothelial cell morphology, cell loss and basement membrane guttae. Ultrastructural analysis shows the predominant defect as dilated endoplasmic reticulum (ER), suggesting ER stress and unfolded protein response (UPR) activation. Immunohistochemistry, western blotting, quantitative reverse transcriptase polymerase chain reaction and terminal deoxynucleotidyl transferase 2-deoxyuridine, 5-triphosphate nick end-labeling analyses support UPR activation and UPR-associated apoptosis in the Col8a2(Q455K/Q455K) mutant corneal endothelium. This study confirms the Q455K substitution in the COL8A2 gene as being sufficient to cause FECD in the first mouse model of this disease and supports the role of the UPR and UPR-associated apoptosis in the pathogenesis of FECD caused by COL8A2 mutations.


Assuntos
Apoptose , Colágeno Tipo VIII/genética , Modelos Animais de Doenças , Endotélio Corneano/metabolismo , Distrofia Endotelial de Fuchs/patologia , Animais , Endotélio Corneano/patologia , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Mutação , Desnaturação Proteica
17.
Exp Eye Res ; 96(1): 147-56, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22200487

RESUMO

Retinal vascular development is a complex process that is not yet fully understood. The majority of research in this area has focused on astrocytes and the template they form in the inner retina, which precedes endothelial cells in the mouse retina. In humans and dogs, however, astrocyte migration follows behind development of blood vessels, suggesting that other cell types may guide this process. One such cell type is the ganglion cell, which differentiates before blood vessel formation and lies adjacent to the primary retinal vascular plexus. The present study investigated the potential role played by ganglion cells in vascular development using Math5(-/-) mice. It has previously been reported that Math5 regulates the differentiation of ganglion cells and Math5(-/-) mice have a 95% reduction in these cells. The development of blood vessels and glia was investigated using Griffonia simplicifolia isolectin B4 labeling and GFAP immunohistochemistry, respectively. JB-4 analysis demonstrated that the hyaloid vessels arose from choriovitreal vessels adjacent to the optic nerve area. As previously reported, Math5(-/-) mice had a rudimentary optic nerve. The primary retinal vessels did not develop post-natally in the Math5(-/-) mice, however, branches of the hyaloid vasculature eventually dove into the retina and formed the inner retinal capillary networks. An astrocyte template only formed in some areas of the Math5(-/-) retina. In addition, GFAP(+) Müller cells were seen throughout the retina that had long processes wrapped around the hyaloid vessels. Transmission electron microscopy confirmed Müller cell abnormalities and revealed disruptions in the inner limiting membrane. The present data demonstrates that the loss of ganglion cells in the Math5(-/-) mice is associated with a lack of retinal vascular development.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neuroglia/patologia , Células Ganglionares da Retina/fisiologia , Vasos Retinianos/patologia , Animais , Animais Recém-Nascidos , Técnica Indireta de Fluorescência para Anticorpo , Deleção de Genes , Técnicas de Genotipagem , Proteína Glial Fibrilar Ácida , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Fibras Nervosas/metabolismo , Fibras Nervosas/patologia , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/metabolismo , Vítreo Primário Hiperplásico Persistente/fisiopatologia , Lectinas de Plantas/metabolismo , Neovascularização Retiniana/fisiopatologia , Vasos Retinianos/metabolismo
18.
BMC Dev Biol ; 11: 60, 2011 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-21999428

RESUMO

BACKGROUND: Valuable insights into the complex process of retinal vascular development can be gained using models with abnormal retinal vasculature. Two such models are the recently described mouse lines with mutations in Lama1, an important component of the retinal internal limiting membrane (ILM). These mutants have a persistence of the fetal vasculature of vitreous (FVV) but lack a primary retinal vascular plexus. The present study provides a detailed analysis of astrocyte and vascular development in these Lama1 mutants. RESULTS: Although astrocytes and blood vessels initially migrate into Lama1 mutant retinas, both traverse the peripapillary ILM into the vitreous by P3. Once in the vitreous, blood vessels anastomose with vessels of the vasa hyaloidea propria, part of the FVV, and eventually re-enter the retina where they dive to form the inner and outer retinal capillary networks. Astrocytes continue proliferating within the vitreous to form a dense mesh that resembles epiretinal membranes associated with persistent fetal vasculature and proliferative vitreoretinopathy. CONCLUSIONS: Lama1 and a fully intact ILM are required for normal retinal vascular development. Mutations in Lama1 allow developing retinal vessels to enter the vitreous where they anastomose with vessels of the hyaloid system which persist and expand. Together, these vessels branch into the retina to form fairly normal inner retinal vascular capillary plexi. The Lama1 mutants described in this report are potential models for studying the human conditions persistent fetal vasculature and proliferative vitreoretinopathy.


Assuntos
Membrana Epirretiniana/metabolismo , Laminina/genética , Mutação , Vasos Retinianos/crescimento & desenvolvimento , Corpo Vítreo/irrigação sanguínea , Animais , Membrana Epirretiniana/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Vasos Retinianos/embriologia , Vitreorretinopatia Proliferativa/embriologia , Vitreorretinopatia Proliferativa/genética , Vitreorretinopatia Proliferativa/metabolismo , Corpo Vítreo/embriologia , Corpo Vítreo/crescimento & desenvolvimento
19.
J Cell Sci ; 124(Pt 4): 523-31, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21266465

RESUMO

Phagocytosis of the shed outer segment discs of photoreceptors is a major function of the retinal pigmented epithelium (RPE). We demonstrate for the first time that ßA3/A1-crystallin, a major structural protein of the ocular lens, is expressed in RPE cells. Further, by utilizing the Nuc1 rat, in which the ßA3/A1-crystallin gene is mutated, we show that this protein is required by RPE cells for proper degradation of outer segment discs that have been internalized in phagosomes. We also demonstrate that in wild-type RPE, ßA3/A1-crystallin is localized to the lysosomes. However, in the Nuc1 RPE, ßA3/A1-crystallin fails to translocate to the lysosomes, perhaps because misfolding of the mutant protein masks sorting signals required for proper trafficking. The digestion of phagocytized outer segments requires a high level of lysosomal enzyme activity, and cathepsin D, the major enzyme responsible for proteolysis of the outer segments, is decreased in mutant RPE cells. Interestingly, our results also indicate a defect in the autophagy process in the Nuc1 RPE, which is probably also linked to impaired lysosomal function, because phagocytosis and autophagy might share common mechanisms in degradation of their targets. ßA3/A1-crystallin is a novel lysosomal protein in RPE, essential for degradation of phagocytosed material.


Assuntos
Cristalinas/genética , Mutação , Fagossomos/genética , Epitélio Pigmentado da Retina/metabolismo , Animais , Cristalinas/metabolismo , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Ratos , Ratos Sprague-Dawley , Epitélio Pigmentado da Retina/ultraestrutura
20.
Ophthalmology ; 118(3): 548-52, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20920828

RESUMO

PURPOSE: This article describes the first retinal histopathologic findings in a patient with Susac's syndrome (SS). DESIGN: Observational case report. PARTICIPANT: A 51-year-old white woman diagnosed with SS. METHODS: Eyes from a 51-year-old white woman diagnosed with SS were obtained at autopsy. One retina was dissected and processed for adenosine diphosphatase (ADPase) flat embedding. Selected areas were processed further for transmission electron microscopy. MAIN OUTCOME MEASURES: Histopathologic examination using ADPase flat-embedding technique. RESULTS: There were vaso-occlusive changes in the retinal periphery resulting in small areas of capillary dropout. Cross-sections demonstrated serous filled spaces between the retinal blood vessels and the internal limiting membrane. Lumens adjacent to these spaces appeared compressed and sometimes closed, but without thrombosis. Decreased ADPase activity in some peripheral blood vessels suggested endothelial cell dysfunction and vaso-occlusion. In the optic nerve head, numerous corpora amylacea were observed in the vicinity of capillaries with thickened walls and narrow lumens. Transmission electron microscopy demonstrated thickened and amorphous vascular basal lamina and open endothelial cell junctions in some retinal blood vessels. CONCLUSIONS: The serous deposits with compression of retinal vessel lumens observed histologically probably represent the so-called string of pearls described clinically in SS. Chronic extension of these serous deposits along the vessel wall possibly are the cause of retinal arterial wall plaques as described by Gass and other investigators. In the optic nerve head, corpora amylacea are probably a result of microinfarcts resulting from optic nerve head capillary angiopathy. Accumulation of amorphous material in the basal lamina, loss of viable endothelial cells, and capillary dropout suggest that SS may be an endotheliopathy.


Assuntos
Disco Óptico/ultraestrutura , Doenças do Nervo Óptico/diagnóstico , Doenças Retinianas/diagnóstico , Vasos Retinianos/ultraestrutura , Síndrome de Susac/diagnóstico , Apirase/metabolismo , Feminino , Humanos , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Disco Óptico/enzimologia , Doenças do Nervo Óptico/enzimologia , Doenças Retinianas/enzimologia , Vasos Retinianos/enzimologia
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