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1.
Invest Ophthalmol Vis Sci ; 62(12): 8, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34505865

RESUMO

Purpose: Oxidative stress is a major factor underlying many neurodegenerative diseases. However, antioxidant therapy has had mixed results, possibly because of its indiscriminate activity. The purpose of our study was to determine if the human OXR1 (hOXR1) antioxidant regulatory gene could protect neurons from oxidative stress and delay photoreceptor cell death. Methods: The cone-like 661W cell line was transfected to stably express the hOXR1 gene. Oxidative stress was induced by the addition of hydrogen peroxide (H2O2). Intracellular levels of reactive oxygen species (ROS), caspase cleavage, and cellular resistance to oxidative stress were determined and compared between the control and hOXR1 cells. For in vivo analysis, AAV8-hOXR1 was injected subretinally into the rd1 mouse model of retinal degeneration. Functional and structural integrity of the photoreceptors were assessed using electroretinography (ERG), histology, and immunofluorescence analysis. Results: Expression of hOXR1 increased cellular resistance and reduced ROS levels and caspase cleavage in the 661W cell line after H2O2-induced oxidative stress. Subretinal injection of AAV8-hOXR1 in the rd1 mice improved their photoreceptor light response, expression and localization of photoreceptor-specific proteins, and delayed retinal degeneration. Conclusions: Our results suggest that OXR1 is a potential therapy candidate for retinal degeneration. Because OXR1 targets oxidative stress, a common feature of many retinal degenerative diseases, it should be of therapeutic value to multiple retinal degenerative diseases.


Assuntos
Regulação da Expressão Gênica , Terapia Genética/métodos , Proteínas Mitocondriais/genética , Estresse Oxidativo , RNA/genética , Retina/patologia , Degeneração Retiniana/terapia , Animais , Morte Celular , Células Cultivadas , Modelos Animais de Doenças , Humanos , Camundongos , Proteínas Mitocondriais/biossíntese , Células Fotorreceptoras de Vertebrados , Espécies Reativas de Oxigênio/metabolismo , Retina/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo
2.
FASEB J ; 35(9): e21802, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34383984

RESUMO

Mutations in transcription factors often exhibit pleiotropic effects related to their complex expression patterns and multiple regulatory targets. One such mutation in the zinc finger homeobox 3 (ZFHX3) transcription factor, short circuit (Sci, Zfhx3Sci/+ ), is associated with significant circadian deficits in mice. However, given evidence of its retinal expression, we set out to establish the effects of the mutation on retinal function using molecular, cellular, behavioral and electrophysiological measures. Immunohistochemistry confirms the expression of ZFHX3 in multiple retinal cell types, including GABAergic amacrine cells and retinal ganglion cells including intrinsically photosensitive retinal ganglion cells (ipRGCs). Zfhx3Sci/+ mutants display reduced light responsiveness in locomotor activity and circadian entrainment, relatively normal electroretinogram and optomotor responses but exhibit an unexpected pupillary reflex phenotype with markedly increased sensitivity. Furthermore, multiple electrode array recordings of Zfhx3Sci/+ retina show an increased sensitivity of ipRGC light responses.


Assuntos
Ritmo Circadiano/fisiologia , Proteínas de Homeodomínio/metabolismo , Retina/metabolismo , Células Amácrinas/metabolismo , Animais , Luz , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estimulação Luminosa/métodos , Células Ganglionares da Retina/metabolismo , Visão Ocular/fisiologia
3.
Zoolog Sci ; 38(4): 326-331, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34342953

RESUMO

The adult lamprey retina has two types of photoreceptor cells, short and long photoreceptor cells, which are equivalent to rods and cones of other vertebrates. In contrast, the retina of lamprey larvae only contains a single type of photoreceptor cell, which appears to correspond to the short photoreceptor cell. However, the developmental pattern of the long photoreceptor cell is unknown. Previously, we reported that antibodies against rhodopsin and iodopsin (the chicken red cone opsin) could discriminate between the outer segments of short and long photoreceptor cells, respectively, in the retina of adult Japanese river lamprey (Lethenteron camtschaticum). Here, we immunohistochemically investigate the appearance of long photoreceptor cells in the larval and adult retinas of the Far Eastern brook lamprey (Lethenteron reissneri), which is a close relative of the Japanese river lamprey, by using anti-iodopsin antibody. We found that iodopsin immunoreactivity was localized not only in the adult retina but also in the larval retina. Moreover, we examined the immunohistochemical localization of signal transduction molecules, such as transducin and arrestin, in the iodopsin-immunoreactive cells of the larval retina. The iodopsin-immunoreactive cells also contained both transducin and arrestin, suggesting that long photoreceptor cells are already functional in the larval stage before the acquisition of visual function. Our results suggest that the iodopsin-immunoreactive cells may be related to not only cone vision in the adult but also photoreception in the larval lamprey.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Imuno-Histoquímica , Lampreias/metabolismo , Células Fotorreceptoras/fisiologia , Animais , Lampreias/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento , Larva/metabolismo , Retina/crescimento & desenvolvimento , Retina/metabolismo
4.
Invest Ophthalmol Vis Sci ; 62(10): 9, 2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34374743

RESUMO

Purpose: Retinal neovascularization is a severe pathological process leading to irreversible blindness. This study aims to identify the altered metabolites and their related pathways that are involved in retinal neovascularization. Methods: To reveal the global metabolomic profile change in the retinal neovascularization process, an untargeted metabolomics analysis of oxygen-induced retinopathy (OIR) mice retinas was carried out first, followed by the validation of amino acids and their derivatives through a targeted metabolomics analysis. The involved pathways were predicted by bioinformatic analysis. Results: By untargeted metabolomics, a total of 58 and 49 metabolites altered significantly in OIR retinas under cationic and anionic modes, respectively. By bioinformatics analysis, "ABC transporters," "central carbon metabolism in cancer." and "alanine, aspartate, and glutamate metabolism" were the most enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with the changed metabolites. By targeted metabolomics, no significant change was found in the assessed amino acids and their derivatives at postnatal day (P) 12, whereas significantly altered amino acids and their derivatives were recognized at P13, P17, and P42 in OIR retinas. Conclusions: The metabolomic profile was significantly altered in the neovascularized retinas. In particular, numerous amino acids and their derivatives were significantly changed in OIR retinas. These altered metabolites, together with their associated pathways, might be involved in the pathogenesis of retinal neovascular diseases.


Assuntos
Metabolômica/métodos , Retina/metabolismo , Neovascularização Retiniana/metabolismo , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Oxigênio/toxicidade , Retina/efeitos dos fármacos , Retina/patologia , Neovascularização Retiniana/induzido quimicamente , Neovascularização Retiniana/patologia
5.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445296

RESUMO

To investigate the retinal proteins associated with primary and secondary retinal ganglion cell (RGC) degeneration and explore their molecular pathways, SWATH label-free and target-based mass spectrometry was employed to identify the proteomes in various retinal locations in response to localized optic nerve injury. Unilateral partial optic nerve transection (pONT) was performed on adult Wistar rats and their retinas were harvested 2 weeks later. To confirm the separation of primary and secondary RGC degeneration, immunohistochemistry of RNA binding protein with multiple splicing (RBPMS) and glial fibrillary acidic protein (GFAP) was performed on retinal whole-mounts. Retinal proteomes in the temporal and nasal quadrants were evaluated with high resolution hybrid quadrupole time-of-flight mass spectrometry (QTOF-MS), and SWATH-based acquisition, and their expression was compared to the corresponding retinal quadrant in contralateral control eyes and further validated by multiple reaction monitoring mass spectrometry (MRM-MS). A total of 3641 proteins (FDR < 1%) were identified using QTOF-MS. The raw data are available via ProteomeXchange with the identifier PXD026783. Bioinformatics data analysis showed that there were 37 upregulated and 25 downregulated proteins in the temporal quadrant, whereas 20 and five proteins were upregulated and downregulated, respectively, in the nasal quadrant, respectively (n = 4, p < 0.05; fold change ≥ 1.4-fold or ≤0.7). Six proteins were regulated in both the temporal and the nasal quadrants, including CLU, GFAP, GNG5, IRF2BPL, L1CAM, and CPLX1. Linear regression analysis indicated a strong association between the data obtained by means of SWATH-MS and MRM-MS (temporal, R2 = 0.97; nasal, R2 = 0.96). Gene ontology analysis revealed statistically significant changes in the biological processes and cellular components of primary RGC degeneration. The majority of the significant changes in structural, signaling, and cell death proteins were associated with the loss of RGCs in the area of primary RGC degeneration. The combined use of SWATH-MS and MRM-MS methods detects and quantifies regional changes of retinal protein expressions after localized injury. Future investigation with this integrated approach will significantly increase the understanding of diverse processes of progressive RGC degeneration from a proteomic prospective.


Assuntos
Proteínas do Olho/metabolismo , Degeneração Retiniana/metabolismo , Células Ganglionares da Retina/metabolismo , Animais , Proteínas do Olho/análise , Espectrometria de Massas/métodos , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Traumatismos do Nervo Óptico/complicações , Traumatismos do Nervo Óptico/metabolismo , Traumatismos do Nervo Óptico/patologia , Proteoma/análise , Proteoma/metabolismo , Proteômica/métodos , Ratos , Ratos Wistar , Retina/química , Retina/metabolismo , Degeneração Retiniana/etiologia , Degeneração Retiniana/patologia , Células Ganglionares da Retina/patologia
6.
Exp Eye Res ; 210: 108715, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34343570

RESUMO

OBJECTIVES: Cone photoreceptor transplantation is a potential treatment for macular diseases. The optimal conditions for cone transplantation are poorly understood, partly because of the scarcity of cones in donor mice. To facilitate allogeneic cone photoreceptor transplantation studies in mice, we aimed to create and characterize a donor mouse model containing a cone-rich retina with a cone-specific enhanced green fluorescent protein (EGFP) reporter. METHODS: We generated OPN1LW-EGFP/NRL-/- mice by crossing NRL-/- and OPN1LW-EGFP mice. We characterized the anatomical phenotype of OPN1LW-EGFP/NRL-/- mice using multimodal confocal scanning laser ophthalmoscopy (cSLO) imaging, immunohistology, and transmission electron microscopy. We evaluated retinal function using electroretinography (ERG), including 465 and 525 nm chromatic stimuli. Retinal sheets and cell suspensions from OPN1LW-EGFP/NRL-/- mice were transplanted subretinally into immunodeficient Rd1 mice. RESULTS: OPN1LW-EGFP/NRL-/- retinas were enriched with OPN1LW-EGFP+ and S-opsin+ cone photoreceptors in a dorsal-ventral distribution gradient. Cone photoreceptors co-expressing OPNL1W-EGFP and S-opsin significantly increased in OPN1LW-EGFP/NRL-/- compared to OPN1LW-EGFP mice. Temporal dynamics of rosette formation in the OPN1LW-EGFP/NRL-/- were similar as the NRL-/- with peak formation at P15. Rosettes formed preferentially in the ventral retina. The outer retina in P35 OPN1LW-EGFP/NRL-/- was thinner than NRL-/- controls. The OPN1LW-EGFP/NRL-/- ERG response amplitudes to 465 nm stimulation were similar to, but to 535 nm stimulation were lower than, NRL-/- controls. Three months after transplantation, the suspension grafts showed greater macroscopic degradation than sheet grafts. Retinal sheet grafts from OPN1LW-EGFP/NRL-/- mice showed greater S-opsin + cone survival than suspension grafts from the same strain. CONCLUSIONS: OPN1LW-EGFP/NRL-/- retinae were enriched with S-opsin+ photoreceptors. Sustained expression of EGFP facilitated the longitudinal tracking of transplanted donor cells. Transplantation of cone-rich retinal grafts harvested prior to peak rosette formation survived and differentiated into cone photoreceptor subtypes. Photoreceptor sheet transplantation may promote greater macroscopic graft integrity and S-opsin+ cone survival than cell suspension transplantation, although the mechanism underlying this observation is unclear at present. This novel cone-rich reporter mouse strain may be useful to study the influence of graft structure on cone survival.


Assuntos
Transplante de Células , Células Fotorreceptoras Retinianas Cones/transplante , Degeneração Retiniana/cirurgia , Animais , Linhagem Celular , Opsinas dos Cones/metabolismo , Eletrorretinografia , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Oftalmoscopia , Retina/metabolismo , Retina/fisiopatologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/fisiopatologia , Opsinas de Bastonetes/metabolismo , Doadores de Tecidos , Transplante Homólogo
7.
FASEB J ; 35(9): e21880, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34449091

RESUMO

In vertebrates, retinal rod and cone photoreceptor cells rely significantly on glycolysis. Lactate released from photoreceptor cells fuels neighboring retinal pigment epithelium cells and Müller glial cells through oxidative phosphorylation. To understand this highly heterogeneous metabolic environment around photoreceptor cells, single-cell analysis is needed. Here, we visualized cellular AMP-activated protein kinase (AMPK) activity and ATP levels in the retina by two-photon microscopy. Transgenic mice expressing a hyBRET-AMPK biosensor were used for measuring the AMPK activity. GO-ATeam2 transgenic mice were used for measuring the ATP level. Temporal metabolic responses were successfully detected in the live retinal explants upon drug perfusion. A glycolysis inhibitor, 2-deoxy-d-glucose (2-DG), activated AMPK and reduced ATP. These effects were clearly stronger in rods than in cones. Notably, rod AMPK and ATP started to recover at 30 min from the onset of 2-DG perfusion. Consistent with these findings, ex vivo electroretinogram recordings showed a transient slowdown in rod dim flash responses during a 60-min 2-DG perfusion, whereas cone responses were not affected. Based on these results, we propose that cones surrounded by highly glycolytic rods become less dependent on glycolysis, and rods also become less dependent on glycolysis within 60 min upon the glycolysis inhibition.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Glicólise/fisiologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Células Ependimogliais/metabolismo , Luz , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação Oxidativa , Fótons , Retina/metabolismo
8.
Molecules ; 26(16)2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34443622

RESUMO

Orexins are two neuropeptides synthesised mainly in the brain lateral hypothalamic area. The orexinergic system provides arousal-dependent cues for a plethora of brain centres, playing a vital role in feeding behaviour, regulation of the sleep-wake cycle and circadian rhythms. Recently, orexins were found to be produced in the retina of an eye; however, their content in the vitreous body and possible daily pattern of expression have not yet been explored. In this manuscript, we describe the development and validation of a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method designed for quantitative bioanalysis of orexin in the rat vitreous body. Orexin was extracted from vitreous body samples with a water:acetonitrile:formic acid (80:20:0.1; v/v/v) mixture followed by vortexing and centrifuging. Separation was performed on a reverse-phase HPLC column under gradient conditions. Orexin was analysed via multiple-reaction monitoring (MRM) in the positive electrospray mode. The total analysis time for each sample was less than 5.0 min. Once the method was fully optimised, it was then validated, following the 2018 FDA guidance on bioanalytical method validations. The calibration curves for orexin (1-500 ng/mL) were constructed using a linear regression with a 1/x2 weighting. The lower limit of quantitation for orexin was 1.0 pg/mL for the vitreous body. Intra-day and inter-day estimates of accuracy and precision were within 10% of their nominal values, indicating that the method is reliable for quantitation of orexin in the rat vitreous body. From the physiological perspective, our results are the first to show daily rhythm of orexin synthesis by the retina with possible implications on the circadian regulation of vision.


Assuntos
Cromatografia Líquida , Ritmo Circadiano , Orexinas/metabolismo , Retina/metabolismo , Espectrometria de Massas em Tandem , Corpo Vítreo/metabolismo , Animais , Calibragem , Modelos Lineares , Masculino , Ratos
9.
FASEB J ; 35(9): e21846, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34405458

RESUMO

Myopia (short-sightedness), usually caused by excessive elongation of the eye during development, has reached epidemic proportions worldwide. In animal systems including the chicken model, several treatments have been shown to inhibit ocular elongation and experimental myopia. Although diverse in their apparent mechanism of action, each one leads to a reduction in the rate of ocular growth. We hypothesize that a defined set of retinal molecular changes may underlie growth inhibition, irrespective of the treatment agent used. Accordingly, across five well-established but diverse methods of inhibiting myopia, significant overlap is seen in the retinal transcriptome profile (transcript levels and alternative splicing events) in chicks when analyzed by RNA-seq. Within the two major pathway networks enriched during growth inhibition, that of cell signaling and circadian entrainment, transcription factors form the largest functional grouping. Importantly, a large percentage of those genes forming the defined retinal response are downstream targets of the transcription factor EGR1 which itself shows a universal response to all five growth-inhibitory treatments. This supports EGR1's previously implicated role in ocular growth regulation. Finally, by contrasting our data with human linkage and GWAS studies on refractive error, we confirm the applicability of our study to the human condition. Together, these findings suggest that a universal set of transcriptome changes, which sit within a well-defined retinal network that cannot be bypassed, is fundamental to growth regulation, thus paving a way for designing novel targets for myopia therapies.


Assuntos
Olho/crescimento & desenvolvimento , Olho/metabolismo , Redes Reguladoras de Genes , Miopia/genética , Miopia/prevenção & controle , Transcriptoma , Processamento Alternativo/efeitos dos fármacos , Animais , Atropina/farmacologia , Galinhas , Ritmo Circadiano/efeitos dos fármacos , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Olho/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Janus Quinases/metabolismo , Masculino , Modelos Biológicos , Ácidos Fosfínicos/farmacologia , Pirenzepina/farmacologia , Piridinas/farmacologia , Reprodutibilidade dos Testes , Retina/efeitos dos fármacos , Retina/crescimento & desenvolvimento , Retina/metabolismo , Fatores de Transcrição STAT/metabolismo , Tetra-Hidronaftalenos/farmacologia , Fatores de Tempo , Transcriptoma/efeitos dos fármacos
10.
Elife ; 102021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34252023

RESUMO

Organoids derived from pluripotent stem cells promise the solution to current challenges in basic and biomedical research. Mammalian organoids are however limited by long developmental time, variable success, and lack of direct comparison to an in vivo reference. To overcome these limitations and address species-specific cellular organization, we derived organoids from rapidly developing teleosts. We demonstrate how primary embryonic pluripotent cells from medaka and zebrafish efficiently assemble into anterior neural structures, particularly retina. Within 4 days, blastula-stage cell aggregates reproducibly execute key steps of eye development: retinal specification, morphogenesis, and differentiation. The number of aggregated cells and genetic factors crucially impacted upon the concomitant morphological changes that were intriguingly reflecting the in vivo situation. High efficiency and rapid development of fish-derived organoids in combination with advanced genome editing techniques immediately allow addressing aspects of development and disease, and systematic probing of impact of the physical environment on morphogenesis and differentiation.


Assuntos
Células-Tronco Embrionárias/citologia , Organogênese , Organoides/citologia , Retina/citologia , Animais , Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Humanos , Morfogênese , Organoides/metabolismo , Oryzias , Células-Tronco Pluripotentes/fisiologia , Retina/crescimento & desenvolvimento , Retina/metabolismo , Peixe-Zebra
11.
Int J Mol Sci ; 22(12)2021 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-34205419

RESUMO

Age-related macular degeneration (AMD) is one of the main causes of deterioration in vision in adults aged 55 and older. In spite of therapies, the progression of the disease is often observed without reverse vision quality. In the present study, we explored whether, in undifferentiated ARPE-19 retinal cells, a disruption of the VEGF receptors (VEGF-R)/caveolin-1 (Cav-1)/protein kinases pathway could be a target for counteracting VEGF secretion. We highlight that Resvega®, a combination of omega-3 fatty acids with an antioxidant, resveratrol, inhibits VEGF-A secretion in vitro by disrupting the dissociation of the VEGF-R2/Cav-1 complex into rafts and subsequently preventing MAPK activation. Moreover, DNA ChIP analysis reveals that this combination prevents the interaction between AP-1 and vegf-a and vegf-r2 gene promoters. By these pathways, Resvega could present a potential interest as nutritional complementation against AMD.


Assuntos
Caveolina 1/metabolismo , Degeneração Macular/prevenção & controle , Retina/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Retina/metabolismo , Fator de Transcrição AP-1/antagonistas & inibidores
12.
Methods Mol Biol ; 2319: 111-117, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331249

RESUMO

The retina offers a unique opportunity to directly visualize blood vessels in vivo noninvasively. Over the past few decades, several new imaging techniques have been adapted to study the retinal vasculature in the laboratory in animal models and in the clinic in human subjects. High-contrast, finely detailed fundus images can be acquired by confocal scanning laser ophthalmoscopy (cSLO). With fluorescein angiography (FA), the retinal microcirculation can be visualized. High-resolution spectral-domain optical coherence tomography (SD-OCT) is able to acquire cross-section images resolving the microarchitecture of the retina, similar to histology. The techniques and protocols for acquiring cSLO, FA, and SD-OCT imaging of the retinal vasculature and morphology in the rodent are described.


Assuntos
Angiofluoresceinografia/métodos , Oftalmoscopia/métodos , Retina/diagnóstico por imagem , Vasos Retinianos/diagnóstico por imagem , Tomografia de Coerência Óptica/métodos , Animais , Angiofluoresceinografia/instrumentação , Retina/metabolismo , Vasos Retinianos/metabolismo , Tomografia de Coerência Óptica/instrumentação
13.
Int J Nanomedicine ; 16: 4481-4494, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34239300

RESUMO

Purpose: Apatinib (Apa) is a novel anti-vascular endothelial growth factor with the potential to treat diabetic retinopathy (DR); a serious condition leading to visual impairment and blindness. DR treatment relies on invasive techniques associated with various complications. Investigating topical routes for Apa delivery to the posterior eye segment is thus promising but also challenging due to ocular barriers. Hence, the study objective was to develop Apa-loaded bovine serum albumin nanoparticles (Apa-BSA-NPs) coated with hyaluronic acid (HA); a natural polymer possessing unique mucoadhesive and viscoelastic features with the capacity to actively target CD44 positive retinal cells, for topical administration in DR. Methods: Apa-BSA-NPs were prepared by desolvation using glutaraldehyde for cross-linking. HA-coated BSA-NPs were also prepared and HA: NPs ratio optimized. Nanoparticles were characterized for colloidal properties, entrapment efficiency (EE%), in vitro drug release and mucoadhesive potential. In vitro cytotoxicity on rabbit corneal epithelial cells (RCE) was assessed using MTT assay, while efficacy was evaluated in vivo in a diabetic rat model by histopathological examination of the retina by light and transmission electron microscopy. Retinal accumulation of fluorescently labeled BSA-NP and HA-BSA-NP was assessed using confocal microscope scanning. Results: Apa-HA-BSA-NPs prepared under optimal conditions showed size, PdI and zeta potential: 222.2±3.56 nm, 0.221±0.02 and -37.3±1.8 mV, respectively. High EE% (69±1%), biphasic sustained release profile with an initial burst effect and mucoadhesion was attained. No evidence of cytotoxicity was observed on RCE cells. In vivo histopathological studies on DR rat model revealed alleviated retinal micro- and ultrastructural changes in the topical HA-Apa-BSA-NP treated eyes with normal basement membrane and retinal thickness comparable to normal control and intravitreally injected nanoparticles. Improved retinal accumulation for HA-BSA-NP was also observed by confocal microscopy. Conclusion: Findings present HA-Apa-BSA-NPs as a platform for enhanced topical therapy of DR overcoming the devastating ocular complications of the intravitreal route.


Assuntos
Retinopatia Diabética/metabolismo , Portadores de Fármacos/química , Ácido Hialurônico/química , Nanopartículas/química , Piridinas/administração & dosagem , Piridinas/química , Soroalbumina Bovina/química , Animais , Retinopatia Diabética/tratamento farmacológico , Liberação Controlada de Fármacos , Tamanho da Partícula , Piridinas/metabolismo , Piridinas/uso terapêutico , Coelhos , Ratos , Retina/metabolismo
14.
FASEB J ; 35(8): e21829, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34314069

RESUMO

Retinal ischemia is a leading cause of irreversible blindness worldwide. Inner retinal dysfunction including loss of retinal ganglion cells is encountered in a number of retinal ischemic disorders. We previously reported administration of two different hypoxia-inducible factor (HIF) inhibitors exerted neuroprotective effects in a murine model of retinal ischemia/reperfusion (I/R) which mimics these disorders, as inner retinal degeneration could be involved in pathological HIF induction. However, this notion needs further investigation. Therefore, in this study, we attempted to use retina-specific Hif-1α conditional knockout (cKO) mice to uncover this notion more clearly under the same condition. Hif-1α cKO mice showed inner retinal neurodegeneration to a lesser extent than control mice. Hif-1α depletion in a murine 661W retinal cell line reduced cell death under pseudohypoxic and hypoxic conditions. Among hypoxia-related genes, the expression of BCL2 19 kDa protein-interacting protein 3 (Bnip3) was substantially upregulated in the inner retinal layer after retinal I/R. In this regard, we further examined Bnip3 depletion in retinal neurons in vitro and in vivo and found the similar neuroprotective effects. Our results support the notion that the HIF-1α/BNIP3 pathway may have a critical role in inner retinal neurodegeneration, which can be linked with the development of new promising therapeutics for inner retinal ischemic disorders.


Assuntos
Hipóxia Celular , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Mitocondriais/fisiologia , Neuroproteção , Retina , Degeneração Retiniana/metabolismo , Animais , Linhagem Celular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Retina/metabolismo , Retina/patologia
15.
Cell Mol Life Sci ; 78(16): 5977-5985, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34230991

RESUMO

Diabetic retinopathy (DR) is the leading cause of vision loss in working adults in developed countries. The disease traditionally classified as a microvascular complication of diabetes is now widely recognized as a neurovascular disorder resulting from disruption of the retinal neurovascular unit (NVU). The NVU comprising retinal neurons, glia and vascular cells coordinately regulates blood flow, vascular density and permeability to maintain homeostasis. Disturbance of the NVU during DR can lead to vision-threatening clinical manifestations. A limited number of signaling pathways have been identified for intercellular communication within the NVU, including vascular endothelial growth factor (VEGF), the master switch for angiogenesis. VEGF inhibitors are now widely used to treat DR, but their limited efficacy implies that other signaling molecules are involved in the pathogenesis of DR. By applying a novel screening technology called comparative ligandomics, we recently discovered secretogranin III (Scg3) as a unique DR-selective angiogenic and vascular leakage factor with therapeutic potential for DR. This review proposes neuron-derived Scg3 as the first diabetes-selective neurovascular regulator and discusses important features of Scg3 inhibition for next-generation disease-targeted anti-angiogenic therapies of DR.


Assuntos
Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Animais , Cromograninas/metabolismo , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Humanos , Neurônios/metabolismo , Neurônios/patologia , Retina/metabolismo , Retina/patologia , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
16.
Int J Mol Sci ; 22(14)2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34298915

RESUMO

Retinal diseases such as age-related macular degeneration (AMD), retinopathy of prematurity (ROP), and diabetic retinopathy (DR) are the leading causes of visual impairment worldwide. There is a critical need to understand the structural and cellular components that play a vital role in the pathophysiology of retinal diseases. One potential component is the family of structural proteins called small leucine-rich proteoglycans (SLRPs). SLRPs are crucial in many fundamental biological processes involved in the maintenance of retinal homeostasis. They are present within the extracellular matrix (ECM) of connective and vascular tissues and contribute to tissue organization and modulation of cell growth. They play a vital role in cell-matrix interactions in many upstream signaling pathways involved in fibrillogenesis and angiogenesis. In this comprehensive review, we describe the expression patterns and function of SLRPs in the retina, including Biglycan and Decorin from class I; Fibromodulin, Lumican, and a Proline/arginine-rich end leucine-rich repeat protein (PRELP) from class II; Opticin and Osteoglycin/Mimecan from class III; and Chondroadherin (CHAD), Tsukushi and Nyctalopin from class IV.


Assuntos
Leucina/metabolismo , Retina/metabolismo , Proteoglicanos Pequenos Ricos em Leucina/metabolismo , Animais , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Humanos
17.
Invest Ophthalmol Vis Sci ; 62(9): 27, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34283211

RESUMO

Purpose: The purpose of this study was to determine whether retinal gap junctions (GJs) via connexin 36 (Cx36, mediating coupling of many retinal cell types) and horizontal cell (HC-HC) coupling, are involved in emmetropization. Methods: Guinea pigs (3 weeks old) were monocularly form deprived (FD) or raised without FD (in normal visual [NV] environment) for 2 days or 4 weeks; alternatively, they wore a -4 D lens (hyperopic defocus [HD]) or 0 D lens for 2 days or 1 week. FD and NV eyes received daily subconjunctival injections of a nonspecific GJ-uncoupling agent, 18-ß-Glycyrrhetinic Acid (18-ß-GA). The amounts of total Cx36 and of phosphorylated Cx36 (P-Cx36; activated state that increases cell-cell coupling), in the inner and outer plexiform layers (IPLs and OPLs), were evaluated by quantitative immunofluorescence (IF), and HC-HC coupling was evaluated by cut-loading with neurobiotin. Results: FD per se (excluding effect of light-attenuation) increased HC-HC coupling in OPL, whereas HD did not affect it. HD for 2 days or 1 week had no significant effect on retinal content of Cx36 or P-Cx36. FD for 4 weeks decreased the total amounts of Cx36 and P-Cx36, and the P-Cx36/Cx36 ratio, in the IPL. Subconjunctival 18-ß-GA induced myopia in NV eyes and increased the myopic shifts in FD eyes, while reducing the amounts of Cx36 and P-Cx36 in both the IPL and OPL. Conclusions: These results suggest that cell-cell coupling via GJs containing Cx36 (particularly those in the IPL) plays a role in emmetropization and form deprivation myopia (FDM) in mammals. Although both FD and 18-ß-GA induced myopia, they had opposite effects on HC-HC coupling. These findings suggest that HC-HC coupling in the OPL might not play a significant role in emmetropization and myopia development.


Assuntos
Conexinas/metabolismo , Emetropia/fisiologia , Junções Comunicantes/metabolismo , Hiperopia/metabolismo , Miopia/metabolismo , Retina/metabolismo , Corpo Vítreo/metabolismo , Animais , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/patologia , Modelos Animais de Doenças , Junções Comunicantes/patologia , Cobaias , Hiperopia/patologia , Hiperopia/fisiopatologia , Miopia/patologia , Miopia/fisiopatologia , Retina/patologia , Retina/fisiopatologia , Privação Sensorial , Corpo Vítreo/patologia
18.
Biomolecules ; 11(7)2021 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203504

RESUMO

Age-related macular degeneration (AMD) causes the degeneration of photoreceptors and retinal cells leading to vision loss in older subjects. Among possible exogenous risk factors, it has been recently proposed that long-term exposure to blue light could aggravate the course of AMD. In the search for therapeutic options, plasma rich in growth factors (PRGF) has been shown to enhance cell antioxidant pathways and protect photoreceptors against the harm produced by blue light, although its mechanism of action remains unknown. One possible mechanism, autophagy, is one of the most conservative cell renewal systems used in eukaryotes to destroy cellular components that have been damaged by some kind of insult. The oxidative stress of exposure to blue light is known to induce cell autophagy. In this study, we examined the combined effects on autophagy of blue light and PRGF in a retinal cell line, ARPE19. In response to treatment with both PRGF and blue light, we detected the modulated expression of autophagy markers such as NF-kB, p62/sqstm1, Atg5, LC3 and Beclin1, and inflammatory markers such as IL1B and IL18. Our findings suggest that PRGF promotes cell autophagy in response to exposure to blue light.


Assuntos
Autofagia/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Luz/efeitos adversos , Estresse Oxidativo/fisiologia , Retina/metabolismo , Adulto , Autofagia/efeitos da radiação , Proteínas Sanguíneas/metabolismo , Proteínas Sanguíneas/efeitos da radiação , Linhagem Celular , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/efeitos da radiação , Masculino , NF-kappa B/sangue , NF-kappa B/efeitos da radiação , Estresse Oxidativo/efeitos da radiação
19.
Biomolecules ; 11(6)2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208233

RESUMO

Age-related macular degeneration (AMD) is a multifactorial disease of unclear etiology. We previously proposed that metabolic adaptations in photoreceptors (PRs) play a role in disease progression. We mimicked these metabolic adaptations in mouse PRs through deletion of the tuberous sclerosis complex (TSC) protein TSC1. Here, we confirm our previous findings by deletion of the other complex protein, namely TSC2, in rod photoreceptors. Similar to deletion of Tsc1, mice with deletion of Tsc2 in rods develop AMD-like pathologies, including accumulation of apolipoproteins, migration of microglia, geographic atrophy, and neovascular pathologies. Subtle differences between the two mouse models, such as a significant increase in microglia activation with loss of Tsc2, were seen as well. To investigate the role of altered glucose metabolism in disease pathogenesis, we generated mice with simulation deletions of Tsc2 and hexokinase-2 (Hk2) in rods. Although retinal lactate levels returned to normal in mice with Tsc2-Hk2 deletion, AMD-like pathologies still developed. The data suggest that the metabolic adaptations in PRs that cause AMD-like pathologies are independent of HK2-mediated aerobic glycolysis.


Assuntos
Degeneração Macular/patologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Ciclo do Ácido Cítrico , Modelos Animais de Doenças , Feminino , Glicólise , Hexoquinase/metabolismo , Hexoquinase/fisiologia , Masculino , Camundongos , Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/fisiologia , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/fisiologia
20.
Exp Eye Res ; 210: 108700, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34245755

RESUMO

Age-related macular degeneration (AMD) is a complex retinal disease with no viable treatment strategy. The causative mechanistic pathway for this disease is not yet clear. Therefore, it is highly warranted to screen effective drugs to treat AMD. Rapamycin are known to inhibit inflammation and has been widely used in the clinic as an immunosuppressant. This study aimed to investigate the protective effect of rapamycin on the AMD retinal degeneration model. The AMD models were established by injection of 35 mg/kg sodium iodate (NaIO3) into the tail vein. Then the treated mice intraperitoneally received rapamycin (2 mg/kg) once a day. The histomorphological analysis showed that rapamycin could inhibit retinal structure damage and apoptosis. Experiments revealed that rapamycin significantly attenuated inflammatory response and oxidative stress. Our experimental results demonstrated that rapamycin has protected the retinal against degeneration induced by NaIO3. The therapeutic effect was more significant after 7 days of treatment. Therefore, our study potentially provides a powerful experimental support for the treatment of AMD.


Assuntos
Modelos Animais de Doenças , Imunossupressores/uso terapêutico , Degeneração Retiniana/prevenção & controle , Epitélio Pigmentado da Retina/efeitos dos fármacos , Sirolimo/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Marcação In Situ das Extremidades Cortadas , Injeções Intraperitoneais , Iodatos/toxicidade , Masculino , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Retina/metabolismo , Retina/patologia , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Rodopsina/metabolismo , cis-trans-Isomerases/metabolismo
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