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1.
Dev Genes Evol ; 230(4): 305-314, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32671457

RESUMO

Crinoids are considered as the most basal extant echinoderms. They retain aboral nervous system with a nerve center, which has been degraded in the eleutherozoan echinoderms. To investigate the evolution of patterning of the nervous systems in crinoids, we examined temporal and spatial expression patterns of three neural patterning-related homeobox genes, six3, pax6, and otx, throughout the development of a feather star Anneissia japonica. These genes were involved in the patterning of endomesodermal tissues instead of the ectodermal neural tissues in the early planktonic stages. In the stages after larval attachment, the expression of these genes was mainly observed in the podia and the oral nervous systems instead of the aboral nerve center. Our results indicate the involvement of these three genes in the formation of oral nervous system in the common ancestor of the echinoderms and suggest that the aboral nerve center is not evolutionally related to the brain of other bilaterians.


Assuntos
Equinodermos/crescimento & desenvolvimento , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição Otx/metabolismo , Fator de Transcrição PAX6/metabolismo , Animais , Padronização Corporal/genética , Equinodermos/genética , Equinodermos/metabolismo , Evolução Molecular , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Larva/genética , Larva/metabolismo , Proteínas do Tecido Nervoso/genética , Sistema Nervoso/crescimento & desenvolvimento , Sistema Nervoso/metabolismo , Neurônios , Fatores de Transcrição Otx/genética , Fator de Transcrição PAX6/genética
2.
Medicine (Baltimore) ; 99(25): e20793, 2020 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-32569224

RESUMO

OBJECTIVE: To study the relationship between single nucleotide polymorphism (SNP) of the 3 primer untranslated region (UTR) variants of the cell fate determination factor Dachshund 1(DACH1) gene and the susceptibility of patients with endometrial cancer (EC). METHODS: Genomic DNA was extracted from the peripheral venous blood of 235 EC patients and 235 healthy controls, and the DACH1 gene rs9285274, rs9529895, rs17088351, and rs59352399 loci were analyzed by Sanger sequencing. Patients progression-free survival (PFS) was recorded after 3 years follow-up from October 2016 to October 2019. RESULTS: Carriers of the C allele of the DACH1 gene rs9529895 locus had a significantly lower risk for EC than T allele carriers (odds ratio = 0.56, 95%confidence interval: 0.38-0.84, P < .01). The correlation between DACH1 gene rs9529895 locus SNP and the risk for EC was affected by age, body mass index, smoking, drinking, and diabetes. Age, and rs9285274, rs9529895, and rs59352399 locus SNP were the best models for predicting the risk for EC. The accuracy rate was 57.02%, and the Cross-validation Consistency was 10/10 (x = 4.33, P = .04). The DACH1 gene rs9529895 locus C allele (TC+CC) carriers had significantly higher PFS than the TT genotype carriers (P = .04). The DACH1 gene was expressed in decreased amounts in the cancer tissues of EC patients, and the DACH1 mRNA expression level in the CC genotype, TC genotype, and TT genotype of rs9529895 locus was also decreased (P = .02). CONCLUSION: DACH1 gene rs9529895 locus SNP is significantly related to the risk for EC and PFS of EC patients. The possible mechanism behind this relationship is that the DACH1 gene rs9529895 locus SNP affects DACH1 expression level.


Assuntos
Neoplasias do Endométrio/genética , Proteínas do Olho/genética , MicroRNAs/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Fatores de Transcrição/genética , Regiões 3' não Traduzidas/genética , Sítios de Ligação/genética , Western Blotting , Estudos de Casos e Controles , Proteínas do Olho/metabolismo , Feminino , Predisposição Genética para Doença/genética , Humanos , Pessoa de Meia-Idade , Intervalo Livre de Progressão , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/metabolismo
3.
PLoS One ; 15(6): e0232111, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32579557

RESUMO

Glaucoma is the second leading cause of irreversible blindness worldwide. Primary open angle glaucoma (POAG), the most common form of glaucoma, is often associated with elevation of intraocular pressure (IOP) due to the dysfunction of trabecular meshwork (TM) tissues. Currently, an ex vivo human anterior segment perfusion cultured system is widely used to study the effects of glaucoma factors and disease modifying drugs on physiological parameters like aqueous humor (AH) dynamics and IOP homeostasis. This system requires the use of freshly enucleated intact human eyes, which are sparsely available at very high cost. In this study, we explored the feasibility of using human donor corneoscleral segments for modeling morphological and biochemical changes associated with POAG. Among the number of corneas donated each year, many are deemed ineligible for transplantation due to stringent acceptance criteria. These ineligible corneoscleral segments were obtained from the Lions Eye Bank, Tampa, Florida. Each human donor anterior corneoscleral segment was dissected into four equal quadrants and cultured for 7 days by treating with the glaucoma factors dexamethasone (Dex) or recombinant transforming growth factor (TGF) ß2 or transduced with lentiviral expression vectors containing wild type (WT) and mutant myocilin. Hematoxylin and Eosin (H&E) staining analysis revealed that the TM structural integrity is maintained after 7 days in culture. Increased TUNEL positive TM cells were observed in corneoscleral quadrants treated with glaucoma factors compared to their respective controls. However, these TUNEL positive cells were mainly confined to the scleral region adjacent to the TM. Treatment of corneoscleral quadrants with Dex or TGFß2 resulted in glaucomatous changes at the TM, which included increased extracellular matrix (ECM) proteins and induction of endoplasmic reticulum (ER) stress. Western blot analysis of the conditioned medium showed an increase in ECM (fibronectin and collagen IV) levels in Dex- or TGFß2-treated samples compared to control. Lentiviral transduction of quadrants resulted in expression of WT and mutant myocilin in TM tissues. Western blot analysis of conditioned medium revealed decreased secretion of mutant myocilin compared to WT myocilin. Moreover, increased ECM deposition and ER stress induction was observed in the TM of mutant myocilin transduced quadrants. Our findings suggest that the ex-vivo cultured human corneoscleral segment model is cost-effective and can be used as a pre-screening tool to study the effects of glaucoma factors and anti-glaucoma therapeutics on the TM.


Assuntos
Dexametasona/farmacologia , Limbo da Córnea/metabolismo , Malha Trabecular/efeitos dos fármacos , Fator de Crescimento Transformador beta2/farmacologia , Apoptose/efeitos dos fármacos , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Glaucoma de Ângulo Aberto/patologia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Limbo da Córnea/citologia , Limbo da Córnea/efeitos dos fármacos , Malha Trabecular/metabolismo , Malha Trabecular/patologia , Regulação para Cima/efeitos dos fármacos
4.
Proc Natl Acad Sci U S A ; 117(26): 15293-15304, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541062

RESUMO

Organisms possess photoperiodic timing mechanisms to detect variations in day length and temperature as the seasons progress. The nature of the molecular mechanisms interpreting and signaling these environmental changes to elicit downstream neuroendocrine and physiological responses are just starting to emerge. Here, we demonstrate that, in Drosophila melanogaster, EYES ABSENT (EYA) acts as a seasonal sensor by interpreting photoperiodic and temperature changes to trigger appropriate physiological responses. We observed that tissue-specific genetic manipulation of eya expression is sufficient to disrupt the ability of flies to sense seasonal cues, thereby altering the extent of female reproductive dormancy. Specifically, we observed that EYA proteins, which peak at night in short photoperiod and accumulate at higher levels in the cold, promote reproductive dormancy in female D. melanogaster Furthermore, we provide evidence indicating that the role of EYA in photoperiodism and temperature sensing is aided by the stabilizing action of the light-sensitive circadian clock protein TIMELESS (TIM). We postulate that increased stability and level of TIM at night under short photoperiod together with the production of cold-induced and light-insensitive TIM isoforms facilitate EYA accumulation in winter conditions. This is supported by our observations that tim null mutants exhibit reduced incidence of reproductive dormancy in simulated winter conditions, while flies overexpressing tim show an increased incidence of reproductive dormancy even in long photoperiod.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Proteínas do Olho/metabolismo , Fotoperíodo , Estações do Ano , Temperatura , Animais , Proteínas de Drosophila/genética , Proteínas do Olho/genética , Regulação da Expressão Gênica/fisiologia , Reprodução
5.
Ren Fail ; 42(1): 463-473, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32408786

RESUMO

Objective: This report was designed to assess the functional role of miR-218/dachshund family transcription factor 1 (DACH1) in diabetic kidney disease (DKD) and investigate its possible molecular mechanism.Materials and Methods: From the GEO database, we downloaded different datasets for analyzing the expression of miR-218 and DACH1 in DKD. TargetScan was adopted to predict the binding sites between miR-218 and DACH1, which was further verified by dual-luciferase reporter assays. The renal proximal tubule cells (HK-2) treated with high glucose (HG) were used as an in vitro model. QRT-PCR and western blot were used to determine the expression of DACH1 and other relative factors. Cell counting kit-8 and flow cytometer were applied to detect cell viability and apoptosis. The levels of inflammatory cytokines were determined by an ELISA assay.Results: A prominent raise of miR-218 was observed in DKD through bioinformatics analysis, which was further confirmed in the HG-induced model. DACH1 is a target of miR-218. miR-218 reduced cell viability and induced apoptosis by negatively regulating DACH1. Moreover, upregulating miR-218 in HG models increased the concentrations of pro-inflammatory cytokines TNF-α and IL-1ß, reduced the level of anti-inflammatory cytokine IL-10, and promoted the epithelial-mesenchymal transition (EMT) process, which is possibly achieved by targeting DACH1. While downregulating miR-218 showed the opposite results.Conclusion: These data demonstrated that, under an in vitro HG environment, miR-218 suppressed the HK-2 cells proliferation, promoted apoptosis, caused an inflammatory response, and facilitated the EMT process largely by targeting DACH1, providing an insight into the therapeutic intervention of DKD.


Assuntos
Apoptose/efeitos dos fármacos , Transição Epitelial-Mesenquimal , Proteínas do Olho/metabolismo , Glucose/farmacologia , MicroRNAs/genética , Fatores de Transcrição/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Proteínas do Olho/genética , Humanos , Inflamação , Rim , Fatores de Transcrição/genética
6.
Proc Natl Acad Sci U S A ; 117(21): 11450-11458, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32385162

RESUMO

Dynamic remodeling of the extracellular matrix affects many cellular processes, either directly or indirectly, through the regulation of soluble ligands; however, the mechanistic details of this process remain largely unknown. Here we propose that type I collagen remodeling regulates the receptor-binding activity of pigment epithelium-derived factor (PEDF), a widely expressed secreted glycoprotein that has multiple important biological functions in tissue and organ homeostasis. We determined the crystal structure of PEDF in complex with a disulfide cross-linked heterotrimeric collagen peptide, in which the α(I) chain segments-each containing the respective PEDF-binding region (residues 930 to 938)-are assembled with an α2α1α1 staggered configuration. The complex structure revealed that PEDF specifically interacts with a unique amphiphilic sequence, KGHRGFSGL, of the type I collagen α1 chain, with its proposed receptor-binding sites buried extensively. Molecular docking demonstrated that the PEDF-binding surface of type I collagen contains the cross-link-susceptible Lys930 residue of the α1 chain and provides a good foothold for stable docking with the α1(I) N-telopeptide of an adjacent triple helix in the fibril. Therefore, the binding surface is completely inaccessible if intermolecular crosslinking between two crosslink-susceptible lysyl residues, Lys9 in the N-telopeptide and Lys930, is present. These structural analyses demonstrate that PEDF molecules, once sequestered around newly synthesized pericellular collagen fibrils, are gradually liberated as collagen crosslinking increases, making them accessible for interaction with their target cell surface receptors in a spatiotemporally regulated manner.


Assuntos
Colágeno Tipo I/metabolismo , Proteínas do Olho/química , Proteínas do Olho/metabolismo , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/metabolismo , Serpinas/química , Serpinas/metabolismo , Sítios de Ligação , Dicroísmo Circular , Colágeno Tipo I/química , Cristalografia por Raios X , Dissulfetos/química , Lisina/química , Simulação de Acoplamento Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Transdução de Sinais , Análise Espaço-Temporal
7.
Invest Ophthalmol Vis Sci ; 61(5): 1, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32392309

RESUMO

Purpose: Mutations in the RS1 gene, which encodes retinoschisin, cause X-linked juvenile retinoschisis, a retinal dystrophy in males. Retinoschisin specifically interacts with the retinal sodium-potassium adenosine triphosphatase (Na/K-ATPase), a transmembrane ion pump. Na/K-ATPases also bind cardiac glycosides, which control the activity of the pump and have been linked to disturbances in retinal homeostasis. In this study, we investigated the crosstalk between retinoschisin and cardiac glycosides at the retinal Na/K-ATPase and the consequences of this interplay on retinal integrity. Methods: The effect of cardiac glycosides (ouabain and digoxin) on the binding of retinoschisin to the retinal Na/K-ATPase was investigated via western blot and immunocytochemistry. Also, the influence of retinoschisin on the binding of cardiac glycosides was analyzed via enzymatic assays, which quantified cardiac glycoside-sensitive Na/K-ATPase pump activity. Moreover, retinoschisin-dependent binding of tritium-labeled ouabain to the Na/K-ATPase was determined. Finally, a reciprocal effect of retinoschisin and cardiac glycosides on Na/K-ATPase localization and photoreceptor degeneration was addressed using immunohistochemistry in retinoschisin-deficient murine retinal explants. Results: Cardiac glycosides displaced retinoschisin from the retinal Na/K-ATPase; however, retinoschisin did not affect cardiac glycoside binding. Notably, cardiac glycosides reduced the capacity of retinoschisin to regulate Na/K-ATPase localization and to protect against photoreceptor degeneration. Conclusions: Our findings reveal opposing effects of retinoschisin and cardiac glycosides on retinal Na/K-ATPase binding and on retinal integrity, suggesting that a fine-tuned interplay between both components is required to maintain retinal homeostasis. This observation provides new insight into the mechanisms underlying the pathological effects of cardiac glycoside treatment on retinal integrity.


Assuntos
Digoxina/metabolismo , Proteínas do Olho/metabolismo , Ouabaína/metabolismo , Retinosquise/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Camundongos Endogâmicos C57BL , Ligação Proteica , Transdução de Sinais
8.
PLoS One ; 15(3): e0229342, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32155173

RESUMO

We aimed to construct a better model for predicting treatment outcomes of anti-vascular endothelial growth factor therapy for neovascular age-related macular degeneration (nAMD) using the concentrations of aqueous humour proteins at baseline and during treatment. From the data of 48 treatment-naïve nAMD eyes that received intravitreal ranibizumab pro re nata for up to 12 months, we used the aqueous humour concentrations of C-X-C motif chemokine ligand 1 (CXCL1), CXCL12, CXCL13, interferon-γ-induced protein 10, monocyte chemoattractant protein 1 (MCP-1), C-C motif chemokine ligand 11, interleukin 6 (IL-6), IL-10, and matrix metalloproteinase 9 (MMP-9). After stepwise regression, multivariate analysis was performed to identify which predictors were significantly associated with best-corrected visual acuity (BCVA) changes and the number of injections. The results demonstrated that besides male sex (ß coefficient = -0.088, P = 0.040) and central retinal thickness (ß coefficient = 0.00051 per µm, P = 0.027), MCP-1 (ß coefficient = 0.44, P < 0.001) and IL-10 (ß coefficient = -0.16, P = 0.033) were significantly correlated with baseline BCVA. Additionally, high MCP-1 at baseline (ß coefficient = -0.20, P = 0.015) and low CXCL13 at baseline (ß coefficient = 0.10, P = 0.0054) were independently associated with better BCVA change at 12 months. High MMP-9 at the first injection (ß coefficient = 0.56, P = 0.01), CXCL12 at the third injection (ß coefficient = 0.10, P = 0.0002), and IL-10 at the third injection (ß coefficient = 1.3, P = 0.001) were predictor variables associated with the increased number of injections. In conclusion, aqueous humour protein concentrations may have predictive abilities of BCVA change over 12 months and the number of injections in pro re nata treatment of exudative nAMD.


Assuntos
Humor Aquoso/metabolismo , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Degeneração Macular/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Ranibizumab/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Idoso , Inibidores da Angiogênese/uso terapêutico , Feminino , Humanos , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Masculino , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Estudos Prospectivos , Resultado do Tratamento
9.
Invest Ophthalmol Vis Sci ; 61(3): 11, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32176262

RESUMO

Purpose: To define remodeling of photoreceptor synaptic terminals and second-order retinal neurons in canine X-linked progressive retinal atrophy 1 caused by a five-nucleotide deletion in the RPGR exon ORF15. Methods: Retinas of normal and mutant dogs were used for gene expression, Western blot, and immunohistochemistry. Cell-specific markers were used to examine disease-dependent retinal remodeling. Results: In mutant retinas, a number of rod axon terminals retract into the outer nuclear layer. This neuritic atrophy preceded significant loss of rods and was evident early in disease. Rod bipolar and horizontal cell processes were found to extend into the outer nuclear layer, where they seemed to form contacts with the spherules of rod photoreceptors. No ectopic rewiring was observed. Because cytoskeletal reorganization was previously shown to underlie photoreceptor axon retraction, we examined normal and mutant retinas for expression of axon guidance receptors ROBO1 and ROBO2, which are known to regulate actin cytoskeleton dynamics. We found that the overall expression of both ROBO1 and ROBO2 is retained at the same level in premature and fully developed normal retinas. However, analysis of predisease and early disease retinas identified markedly decreased levels of ROBO1 in rod spherules compared with controls. In contrast, no differences in ROBO1 signals were noted in cone pedicles in normal and mutant retinas, where ROBO1 levels remained similarly low. Conclusions: Depletion of ROBO1 in rod synaptic terminals correlates with the remodeling of axonal and dendritic processes in the outer retina of dogs with X-linked progressive retinal atrophy 1 and may play a role in the retraction of rod axons.


Assuntos
Doenças do Cão/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores Imunológicos/metabolismo , Degeneração Retiniana/metabolismo , Animais , Orientação de Axônios/fisiologia , Axônios/patologia , Doenças do Cão/genética , Doenças do Cão/patologia , Cães , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Doenças Genéticas Ligadas ao Cromossomo X/veterinária , Microscopia Confocal , Proteínas do Tecido Nervoso/deficiência , Plasticidade Neuronal/fisiologia , Terminações Pré-Sinápticas/patologia , Receptores Imunológicos/deficiência , Retina/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Degeneração Retiniana/veterinária , Neurônios Retinianos/metabolismo , Neurônios Retinianos/patologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia
10.
Invest Ophthalmol Vis Sci ; 61(3): 46, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32207812

RESUMO

Purpose: To scrutinize alterations in cellular interactions and cell signaling in the glaucomatous retina, mouse retinal explants were exposed to elevated pressure. Methods: Retinal explants were prepared from C57bl6 mice and cultivated in a pressure chamber under normotensive (atmospheric pressure + 0 mm Hg), moderately elevated (30 mm Hg), and highly elevated (60 mm Hg) pressure conditions. The expression levels of proteins involved in the formation of tight junctions (zonula occludens 1 [ZO-1], occludin, and claudin-5) and adherens junctions (VE-cadherin and ß-catenin) and in cell-signaling cascades (Cdc42 and activated Cdc42 kinase 1 [ACK1]), as well as the expression levels of the growth-factor receptors platelet-derived growth factor receptor beta and vascular endothelial growth factor receptors 1 and 2 (VEGFR-1, VEGFR-2) and of diverse intracellular proteins (ß-III-tubulin, glial fibrillary acidic protein transcript variant 1, α-smooth muscle actin, vimentin, and von Willebrand factor VIII), were analyzed using immunohistochemistry, western blotting, and quantitative real-time polymerase chain reactions. Results: The retinal explants were well preserved when cultured in the pressure chambers used in this study. The responses to pressure elevation varied among diverse retinal cells. Under elevated pressure, the expression of ZO-1 increased in the large vessels, neuronal cells began to express VEGFR-1, and the Cdc42 expression in the optic nerve head was downregulated. Overall we found significant transcriptional downregulation of VE-cadherin, ß-catenin, VEGFR-1, VEGFR-2, vimentin, Cdc42, and ACK1. Western blotting and immunohistochemistry indicated a loss of VE-cadherin with pressure elevation, whereas the protein levels of ZO-1, occludin, VEGFR-1, and ACK1 increased. Conclusions: The pressure chamber used for cultivating mouse retinal explants can serve as an in vitro model system for investigating molecular alterations in glaucoma. In this system, responses of the entire retinal cells toward elevated pressure with conspicuous changes in the vasculature and the optic nerve head can be seen. In particular, our investigations indicate that changes in the blood-retina barrier and in cellular signaling are induced by pressure elevation.


Assuntos
Junções Aderentes/metabolismo , Proteínas do Olho/metabolismo , Glaucoma/metabolismo , Retina/metabolismo , Junções Íntimas/metabolismo , Animais , Antígenos CD/metabolismo , Pressão Atmosférica , Western Blotting , Caderinas/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Ocludina/metabolismo , Técnicas de Cultura de Órgãos , Proteínas Tirosina Quinases/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteína da Zônula de Oclusão-1/metabolismo , beta Catenina/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo
11.
Invest Ophthalmol Vis Sci ; 61(3): 43, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32207813

RESUMO

Purpose: Although stem cell activity represents a crucial feature in corneal and ocular surface homeostasis, other cells populating this region and the neighboring zones might participate and influence local microenvironment. Mast cells, the long-lived and tissue-sited immune cells, have been previously reported in corneoscleral specimens. Herein, mast cells were investigated in corneoscleral tissues and related to microenvironment protein expression. Methods: Twenty-six (14 male/12 female; older than 60 years) human corneoscleral specimens were sectioned for light and fluorescent immunostaining (CD45, p63, Ck-3/7/12/19, tryptase/AA1, and chymase/CC1). Corneal, limbal, and conjunctival squares were produced for molecular and biochemical analysis. Statistical comparisons were carried out by ANOVA. Results: Toluidine blue staining identified metachromatic intact or degranulated mast cells in the area below the palisades' Vogt (Ck-3/12-positive epithelium and underneath p63 immunoreactivity). Tryptase immunoreactivity was observed close to palisades' Vogt, whereas no specific signal was detected for chymase. Tryptase/AA1 transcripts were quantified in limbal and conjunctival RNA extracts, whereas no specific amplification was detected in corneal ones. Few mediators were overexpressed in limbal extracts with respect to corneal (Neural cell adhesion molecule (NCAM), Intercellular adhesion molecule 3 (ICAM3), Brain-derived Neurotrophic factor (BDNF), and neurotrophin 3 (NT3); P < 0.00083) and conjunctival (NCAM, ICAM3, and NT3; P < 0.05) protein extracts. A trend to an increase was observed for Nerve Growth Factor (NGF) in limbal extracts (P > 0.05). Conclusions: The specific observation of tryptase phenotype and the interesting protein signature of microenvironment (adhesion molecules, growth factors, and neurotrophins), known to partake mast cell behavior, at least in other areas, would provide additional information to better understand this crucial zone in the framework of ocular surface healthiness.


Assuntos
Microambiente Celular/fisiologia , Limbo da Córnea/citologia , Mastócitos/fisiologia , Idoso , Antígenos CD/metabolismo , Western Blotting , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Antígeno CD56/metabolismo , Moléculas de Adesão Celular/metabolismo , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Proteínas do Olho/metabolismo , Feminino , Humanos , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Neurotrofina 3/metabolismo , Análise Serial de Proteínas , Reação em Cadeia da Polimerase em Tempo Real , Doadores de Tecidos
12.
J Endocrinol ; 245(2): 291-300, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32171180

RESUMO

Polycystic ovary syndrome (PCOS), one of the most common female endocrine disorder, is a prevalent cause of infertility. Hyperandrogenism is a key feature in PCOS and is correlated with increased expression of VEGF and cytokines in the ovaries. We have previously shown that pigment epithelium-derived factor (PEDF), an endogenous protein, presents potent anti-angiogenic and anti-inflammatory activities in the ovary and negates the effects of cytokines and VEGF. Additionally, PEDF plays a role in both pathophysiology and treatment of ovarian-hyperstimulation syndrome (OHSS), frequently seen in PCOS patients. We established hyperandrogenic-PCOS models, both in vivo, using mice exposed prenatally to dihydrotestosterone (DHT) and, in vitro, using human primary granulosa cells (hpGCs) and human granulosa cell line (KGN). In PCOS-induced mice, the mRNA levels of I l-6, V egf and Amh were higher than those of control; yet, treatment with rPEDF decreased these levels. Moreover, treating OHSS-induced PCOS-mice with rPEDF alleviated all OHSS symptoms. Stimulation of hpGCs with DHT resulted in downregulation of PEDF mRNA expression, concomitantly with a significant increase in IL-6 and IL-8 mRNAs expression. However, co-stimulation of DHT with rPEDF attenuated the increase in cytokines expression. The anti-inflammatory effect of PEDF was found to be mediated via PPARγ pathway. Our findings suggest that rPEDF treatment may normalize the ovarian angiogenic-inflammatory imbalance, induced by PCOS-associated hyperandrogenism. Moreover, the therapeutic potency of PEDF in preventing OHSS symptomes offers a rationale for using PEDF as novel physiological treatment for PCOS sequels.


Assuntos
Anti-Inflamatórios/metabolismo , Proteínas do Olho/metabolismo , Hiperandrogenismo/metabolismo , Fatores de Crescimento Neural/metabolismo , Síndrome do Ovário Policístico/metabolismo , Serpinas/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Di-Hidrotestosterona , Modelos Animais de Doenças , Feminino , Células da Granulosa/metabolismo , Humanos , Hiperandrogenismo/induzido quimicamente , Hiperandrogenismo/complicações , Camundongos , Ovário/metabolismo , Síndrome do Ovário Policístico/induzido quimicamente
13.
Cancer Res ; 80(9): 1861-1874, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32132110

RESUMO

Skeletal muscle wasting is a devastating consequence of cancer that contributes to increased complications and poor survival, but is not well understood at the molecular level. Herein, we investigated the role of Myocilin (Myoc), a skeletal muscle hypertrophy-promoting protein that we showed is downregulated in multiple mouse models of cancer cachexia. Loss of Myoc alone was sufficient to induce phenotypes identified in mouse models of cancer cachexia, including muscle fiber atrophy, sarcolemmal fragility, and impaired muscle regeneration. By 18 months of age, mice deficient in Myoc showed significant skeletal muscle remodeling, characterized by increased fat and collagen deposition compared with wild-type mice, thus also supporting Myoc as a regulator of muscle quality. In cancer cachexia models, maintaining skeletal muscle expression of Myoc significantly attenuated muscle loss, while mice lacking Myoc showed enhanced muscle wasting. Furthermore, we identified the myocyte enhancer factor 2 C (MEF2C) transcription factor as a key upstream activator of Myoc whose gain of function significantly deterred cancer-induced muscle wasting and dysfunction in a preclinical model of pancreatic ductal adenocarcinoma (PDAC). Finally, compared with noncancer control patients, MYOC was significantly reduced in skeletal muscle of patients with PDAC defined as cachectic and correlated with MEF2c. These data therefore identify disruptions in MEF2c-dependent transcription of Myoc as a novel mechanism of cancer-associated muscle wasting that is similarly disrupted in muscle of patients with cachectic cancer. SIGNIFICANCE: This work identifies a novel transcriptional mechanism that mediates skeletal muscle wasting in murine models of cancer cachexia that is disrupted in skeletal muscle of patients with cancer exhibiting cachexia.


Assuntos
Caquexia/complicações , Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas/metabolismo , Músculo Esquelético/metabolismo , Doenças Musculares/metabolismo , Síndrome de Emaciação/etiologia , Animais , Composição Corporal , Caquexia/metabolismo , Carcinoma Ductal Pancreático/complicações , Carcinoma Ductal Pancreático/metabolismo , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Diafragma/fisiologia , Modelos Animais de Doenças , Regulação para Baixo , Proteínas do Olho/genética , Feminino , Glicoproteínas/deficiência , Glicoproteínas/genética , Xenoenxertos , Humanos , Fatores de Transcrição MEF2/metabolismo , Masculino , Camundongos , Músculo Esquelético/patologia , Atrofia Muscular , Doenças Musculares/etiologia , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/complicações , Neoplasias Pancreáticas/metabolismo , RNA Mensageiro/metabolismo , Regeneração , Corrida , Sarcolema , Síndrome de Emaciação/metabolismo , Síndrome de Emaciação/prevenção & controle
14.
PLoS One ; 15(2): e0227524, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32101556

RESUMO

Experimental autoimmune uveitis (EAU) in rodents recapitulates many features of the disease in humans and has served as a useful tool for the development of therapeutics. A peptide from C-terminus of interferon α1, conjugated to palmitoyl-lysine for cell penetration, denoted as IFNα-C, was tested for its anti-inflammatory properties in ARPE-19 cells, followed by testing in a mouse model of EAU. Treatment with IFNα-C and evaluation by RT-qPCR showed the induction of anti-inflammatory cytokines and chemokine. Inflammatory markers induced by treatment with TNFα were suppressed when IFNα-C was simultaneously present. TNF-α mediated induction of NF-κB and signaling by IL-17A were attenuated by IFNα-C. Differentiated ARPE-19 cells were treated with TNFα in the presence or absence IFNα-C and analyzed by immmunhistochemistry. IFNα-C protected against the disruption integrity of tight junction proteins. Similarly, loss of transepithelial resistance caused by TNFα was prevented by IFNα-C. B10.RIII mice were immunized with a peptide from interphotoreceptor binding protein (IRBP) and treated by gavage with IFNα-C. Development of uveitis was monitored by histology, fundoscopy, SD-OCT, and ERG. Treatment with IFNα-C prevented uveitis in mice immunized with the IRBP peptide. Splenocytes isolated from mice with ongoing EAU exhibited antigen-specific T cell proliferation that was inhibited in the presence of IFNα-C. IFNα-C peptide exhibits anti-inflammatory properties and protects mice against damage to retinal structure and function suggesting that it has therapeutic potential for the treatment of autoimmune uveitis.


Assuntos
Doenças Autoimunes/tratamento farmacológico , Interferon Tipo I/química , Peptídeos/uso terapêutico , Retina/patologia , Uveíte/tratamento farmacológico , Administração Oral , Animais , Doenças Autoimunes/patologia , Doenças Autoimunes/fisiopatologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Impedância Elétrica , Eletrorretinografia , Proteínas do Olho/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-17/metabolismo , Camundongos , NF-kappa B/metabolismo , Retina/efeitos dos fármacos , Retina/fisiopatologia , Proteínas de Ligação ao Retinol/metabolismo , Transdução de Sinais/efeitos dos fármacos , Baço/efeitos dos fármacos , Baço/patologia , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Uveíte/patologia , Uveíte/fisiopatologia
15.
Biochem Soc Trans ; 48(1): 187-197, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32065234

RESUMO

Membrane contact sites (MCSs) are sites where the membranes of two different organelles come into close apposition (10-30 nm). Different classes of proteins populate MCSs including factors that act as tethers between the two membranes, proteins that use the MCSs for their function (mainly lipid or ion exchange), and regulatory proteins and enzymes that can act in trans across the MCSs. The ER-Golgi MCSs were visualized by electron microscopists early in the sixties but have remained elusive for decades due to a lack of suitable methodological approaches. Here we report recent progress in the study of this class of MCSs that has led to the identification of their main morphological features and of some of their components and roles. Among these, lipid transfer proteins and lipid exchange have been the most studied and understood so far. However, many unknowns remain regarding their regulation and their role in controlling key TGN functions such as sorting and trafficking as well as their relevance in physiological and pathological conditions.


Assuntos
Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Rede trans-Golgi/metabolismo , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Olho/metabolismo , Homeostase , Humanos , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Varredura/métodos , Microscopia de Fluorescência/métodos , Fosfatidilinositóis/metabolismo , Transporte Proteico
16.
Proc Natl Acad Sci U S A ; 117(9): 5016-5027, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32071204

RESUMO

During development, neural progenitors change their competence states over time to sequentially generate different types of neurons and glia. Several cascades of temporal transcription factors (tTFs) have been discovered in Drosophila to control the temporal identity of neuroblasts, but the temporal regulation mechanism is poorly understood in vertebrates. Mammalian retinal progenitor cells (RPCs) give rise to several types of neuronal and glial cells following a sequential yet overlapping temporal order. Here, by temporal cluster analysis, RNA-sequencing analysis, and loss-of-function and gain-of-function studies, we show that the Fox domain TF Foxn4 functions as a tTF during retinogenesis to confer RPCs with the competence to generate the mid/late-early cell types: amacrine, horizontal, cone, and rod cells, while suppressing the competence of generating the immediate-early cell type: retinal ganglion cells (RGCs). In early embryonic retinas, Foxn4 inactivation causes down-regulation of photoreceptor marker genes and decreased photoreceptor generation but increased RGC production, whereas its overexpression has the opposite effect. Just as in Drosophila, Foxn4 appears to positively regulate its downstream tTF Casz1 while negatively regulating its upstream tTF Ikzf1. Moreover, retina-specific ablation of Foxn4 reveals that it may be indirectly involved in the synaptogenesis, establishment of laminar structure, visual signal transmission, and long-term maintenance of the retina. Together, our data provide evidence that Foxn4 acts as a tTF to bias RPCs toward the mid/late-early cell fates and identify a missing member of the tTF cascade that controls RPC temporal identities to ensure the generation of proper neuronal diversity in the retina.


Assuntos
Proteínas do Olho/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Neurogênese/fisiologia , Retina/metabolismo , Animais , Proteínas de Ligação a DNA , Drosophila , Proteínas do Olho/genética , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento , Fator de Transcrição Ikaros , Camundongos , Camundongos Knockout para ApoE , Neuroglia/citologia , Neuroglia/metabolismo , RNA-Seq , Retina/citologia , Células Fotorreceptoras Retinianas Cones/classificação , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Análise de Sequência , Fatores de Transcrição
17.
Invest Ophthalmol Vis Sci ; 61(2): 30, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32084266

RESUMO

Purpose: Extracellular vesicles (EVs) contain RNA and protein cargo reflective of the genotype and phenotype of the releasing cell of origin. Adult neural retina EV release, RNA transfer, and proteomic cargo are the focus of this study. Methods: Adult wild-type mouse retinae were cultured and released EV diameters and concentrations quantified using Nanosight. Immunogold transmission electron microscopy (TEM) was used to image EV ultrastructure and marker protein localization. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze retinal cell transcripts present in EVs. Super-resolution microscopy was used to image fluorescent (green) RNA and (red) lipid membrane labeled EVs, released by adult retina, and internalized by isolated retinal cells. Mass spectrometry was used to characterize the proteomes of adult retina and EVs. Results: Adult neural retina released EVs at a rate of 1.42 +/- 0.08 × 108/mL over 5 days, with diameters ranging from 30 to 910 nm. The canonical EV markers CD63 and Tsg101 localized to retinal EVs. Adult retinal and neuronal mRNA species present in both retina and EVs included rhodopsin and the neuronal nuclei marker NeuN. Fluorescently labeled RNA in retinal cells was enclosed in EVs, transported to, and uptaken by co-cultured adult retinal cells. Proteomic analysis revealed 1696 protein species detected only in retinal cells, 957 species shared between retina and EVs, and 82 detected only in EVs. Conclusions: The adult neural retina constitutively releases EVs with molecular cargo capable of intercellular transport and predicted involvement in biological processes including retinal physiology, mRNA processing, and transcription regulation within the retinal microenvironment.


Assuntos
Vesículas Extracelulares/metabolismo , Proteínas do Olho/metabolismo , Neurônios/metabolismo , Transporte Proteico/fisiologia , Transporte de RNA/fisiologia , Retina/metabolismo , Animais , Camundongos , Microscopia Eletrônica de Transmissão , RNA Mensageiro/metabolismo
18.
Invest Ophthalmol Vis Sci ; 61(2): 6, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32031578

RESUMO

Purpose: To determine whether priming of bone marrow mesenchymal stem cells (MSCs) by signals from injured retina, particularly tumor necrosis factor α (TNFα), increase their exosomes' neuroprotective efficacy on retinal ganglion cells (RGCs). Methods: MSCs were primed with retinal cell culture conditioned medium, with or without the TNFα blocker etanercept or TNFα prior to isolation of exosomes. MSC conditioned medium or exosomes were added to rat retinal cultures or human stem cell-derived retinal ganglion cell (hRGC) cultures, and RGC neuroprotective effects were quantified. Luminex assays were used to compare primed versus unprimed exosomes. Results: MSC conditioned medium and exosomes exerted a significant neuroprotective effect on injured rat and hRGC. This effect was significantly increased after MSCs were primed with retinal conditioned medium or TNFα. Blocking of TNFα signaling with etanercept prevented priming-induced RGC neuroprotective efficacy. Priming increased PEDF and VEGF-AA exosomal abundance. Conclusions: MSC exosomes promote RGC survival not just in rodent retinal cultures but also with hRGC. Their efficacy can be further enhanced through TNFα priming with the mechanism of action potentially mediated, at least in part, through increased levels of PEDF and VEGF-AA.


Assuntos
Células-Tronco Mesenquimais/fisiologia , Fármacos Neuroprotetores/farmacologia , Células Ganglionares da Retina/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Células Cultivadas , Etanercepte/farmacologia , Exossomos , Proteínas do Olho/metabolismo , Feminino , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Fatores de Crescimento Neural/metabolismo , Neuroproteção/efeitos dos fármacos , Ratos Sprague-Dawley , Serpinas/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo
19.
Biochem Biophys Res Commun ; 524(3): 542-548, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32014251

RESUMO

ES1 homologs are conserved among prokaryotes and eukaryotes, and the gene expression of ES1 homologs has been confirmed in diverse mammalian tissues. However, the localization and function of mammalian ES1 proteins remain poorly understood. ES1 protein was found specifically expressed in the cone cells of zebrafish and was proposed to contribute to the formation of mega mitochondria. We also observed mega mitochondria in the cone cells of porcine retinas, which raised the question regarding the localization of the porcine ES1. Therefore, in the present study, we aimed to determine the localization of ES1 in porcine retinas. We prepared a rabbit polyclonal antibody against the ES1 C-terminal and performed western blotting analysis and immunoelectron microscopy. The ES1 was found to be localized mainly in the mitochondrial intermembrane space of the porcine retinal cells. Immunopositive signals for ES1 were observed in the mitochondria of almost all retinal cells, and not specifically in cone cells. Our results and the ES1 sequences indicated that the glyoxalase III activity of ES1 might contribute to the stable functionality of the active mitochondria in a protective manner.


Assuntos
Proteínas do Olho/metabolismo , Membranas Mitocondriais/metabolismo , Retina/citologia , Homologia de Sequência de Aminoácidos , Suínos/metabolismo , Sequência de Aminoácidos , Animais , Proteínas do Olho/química , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/ultraestrutura , Retina/ultraestrutura , Solubilidade
20.
EBioMedicine ; 52: 102636, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32028070

RESUMO

BACKGROUND: Neurodegenerative diseases are incurable disorders caused by progressive neuronal cell death. Retinitis pigmentosa (RP) is a blinding neurodegenerative disease that results in photoreceptor death and progresses to the loss of the entire retinal network. We previously found that proteomic analysis of the adjacent vitreous served as way to indirectly biopsy the retina and identify changes in the retinal proteome. METHODS: We analyzed protein expression in liquid vitreous biopsies from autosomal recessive (ar)RP patients with PDE6A mutations and arRP mice with Pde6ɑ mutations. Proteomic analysis of retina and vitreous samples identified molecular pathways affected at the onset of photoreceptor death. Based on affected molecular pathways, arRP mice were treated with a ketogenic diet or metabolites involved in fatty-acid synthesis, oxidative phosphorylation, and the tricarboxylic acid (TCA) cycle. FINDINGS: Dietary supplementation of a single metabolite, ɑ-ketoglutarate, increased docosahexaeonic acid levels, provided neuroprotection, and enhanced visual function in arRP mice. A ketogenic diet delayed photoreceptor cell loss, while vitamin B supplementation had a limited effect. Finally, desorption electrospray ionization mass spectrometry imaging (DESI-MSI) on ɑ-ketoglutarate-treated mice revealed restoration of metabolites that correlated with our proteomic findings: uridine, dihydrouridine, and thymidine (pyrimidine and purine metabolism), glutamine and glutamate (glutamine/glutamate conversion), and succinic and aconitic acid (TCA cycle). INTERPRETATION: This study demonstrates that replenishing TCA cycle metabolites via oral supplementation prolongs retinal function and provides a neuroprotective effect on the photoreceptor cells and inner retinal network. FUNDING: NIH grants [R01EY026682, R01EY024665, R01EY025225, R01EY024698, R21AG050437, P30EY026877, 5P30EY019007, R01EY018213, F30EYE027986, T32GM007337, 5P30CA013696], NSF grant CHE-1734082.


Assuntos
Biópsia Líquida , Proteoma , Proteômica , Degeneração Retiniana/diagnóstico , Degeneração Retiniana/metabolismo , Animais , Morte Celular , Sobrevivência Celular , Cromatografia Líquida , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/deficiência , Suplementos Nutricionais , Modelos Animais de Doenças , Progressão da Doença , Eletrorretinografia , Proteínas do Olho/metabolismo , Feminino , Humanos , Biópsia Líquida/métodos , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Neurônios/patologia , Fosforilação Oxidativa , Linhagem , Fenótipo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Proteômica/métodos , Degeneração Retiniana/etiologia , Degeneração Retiniana/terapia , Espectrometria de Massas em Tandem , Tomografia de Coerência Óptica
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