RESUMO
Glaucoma is one of the most common causes of treatable visual impairment in the developed world, affecting approximately 64 million people worldwide, some of whom will be bilaterally blind from irreversible optic nerve damage. The optic nerve head is a key site of damage in glaucoma where there is fibrosis of the connective tissue in the lamina cribrosa (LC) extracellular matrix. As a ubiquitous second messenger, calcium (Ca2+) can interact with various cellular proteins to regulate multiple physiological processes and contribute to a wide range of diseases, including cancer, fibrosis, and glaucoma. Our research has shown evidence of oxidative stress, mitochondrial dysfunction, an elevated expression of Ca2+ entry channels, Ca2+-dependent pumps and exchangers, and an abnormal rise in cytosolic Ca2+ in human glaucomatous LC fibroblast cells. We have evidence that this increase is dependent on Ca2+ entry channels located in the plasma membrane, and its release is from internal stores in the endoplasmic reticulum (ER), as well as from the mitochondria. Here, we summarize some of the molecular Ca2+-dependent mechanisms related to this abnormal Ca2+-signalling in human glaucoma LC cells, with a view toward identifying potential therapeutic targets for ongoing optic neuropathy.
Assuntos
Glaucoma , Disco Óptico , Humanos , Cálcio/metabolismo , Miofibroblastos/metabolismo , Glaucoma/metabolismo , Disco Óptico/metabolismo , Fibrose , Pressão IntraocularRESUMO
Purpose: Extracellular matrix stiffening is characteristic of both aging and glaucoma, and acts as a promoter and perpetuator of pathological fibrotic remodeling. Here, we investigate the role of a mechanosensitive transcriptional coactivator, Yes-associated protein (YAP), a downstream effector of multiple signaling pathways, in lamina cribrosa (LC) cell activation to a profibrotic, glaucomatous state. Methods: LC cells isolated from glaucomatous human donor eyes (GLC; n = 3) were compared to LC cells from age-matched nonglaucomatous controls (NLC; n = 3) to determine differential YAP expression, protein levels, and proliferation rates. NLC cells were then cultured on soft (4 kPa), and stiff (100 kPa), collagen-1 coated polyacrylamide hydrogel substrates. Quantitative real-time RT-PCR, immunoblotting, and immunofluorescence microscopy were used to measure the expression, activity, and subcellular location of YAP and its downstream targets, respectively. Proliferation rates were examined in NLC and GLC cells by methyl thiazolyl tetrazolium salt assays, across a range of incrementally increased substrate stiffness. Endpoints were examined in the presence or absence of a YAP inhibitor, verteporfin (2 µM). Results: GLC cells show significantly (P < 0.05) increased YAP gene expression and total-YAP protein compared to NLC cells, with significantly increased proliferation. YAP regulation is mechanosensitive, because NLC cells cultured on pathomimetic, stiff substrates (100 kPa) show significantly upregulated YAP gene and protein expression, increased YAP phosphorylation at tyrosine 357, reduced YAP phosphorylation at serine 127, increased nuclear pooling, and increased transcriptional target, connective tissue growth factor. Accordingly, myofibroblastic markers, α-smooth muscle actin (α-SMA) and collagen type I, alpha 1 (Col1A1) are increased. Proliferation rates are elevated on 50 kPa substrates and tissue culture plastic. Verteporfin treatment significantly inhibits YAP-mediated cellular activation and proliferation despite a stiffened microenvironment. Conclusions: These data demonstrate how YAP plays a pivotal role in LC cells adopting a profibrotic and proliferative phenotype in response to the stiffened LC present in aging and glaucoma. YAP provides an attractive and novel therapeutic target, and its inhibition via verteporfin warrants further clinical investigation.
Assuntos
Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Glaucoma/genética , Mecanotransdução Celular/fisiologia , Disco Óptico/metabolismo , Proteínas Proto-Oncogênicas c-yes/genética , Proteínas de Sinalização YAP/genética , Western Blotting , Células Cultivadas , Glaucoma/metabolismo , Glaucoma/patologia , Humanos , Disco Óptico/patologia , Proteínas Proto-Oncogênicas c-yes/biossíntese , RNA/genética , Proteínas de Sinalização YAP/biossínteseRESUMO
Astrocytes within the optic nerve head undergo actin cytoskeletal rearrangement early in glaucoma, which coincides with astrocyte reactivity and extracellular matrix (ECM) deposition. Elevated transforming growth factor beta 2 (TGFß2) levels within astrocytes have been described in glaucoma, and TGFß signaling induces actin cytoskeletal remodeling and ECM deposition in many tissues. A key mechanism by which astrocytes sense and respond to external stimuli is via mechanosensitive ion channels. Here, we tested the hypothesis that inhibition of mechanosensitive channels will attenuate TGFß2-mediated optic nerve head astrocyte actin cytoskeletal remodeling, reactivity, and ECM deposition. Primary optic nerve head astrocytes were isolated from C57BL/6J mice and cell purity was confirmed by immunostaining. Astrocytes were treated with vehicle control, TGFß2 (5 ng/ml), GsMTx4 (a mechanosensitive channel inhibitor; 500 nM), or TGFß2 (5 ng/ml) + GsMTx4 (500 nM) for 48 h. FITC-phalloidin staining was used to assess the formation of f-actin stress fibers and to quantify the presence of crosslinked actin networks (CLANs). Cell reactivity was determined by immunostaining and immunoblotting for GFAP. Levels of fibronectin and collagen IV deposition were also quantified. Primary optic nerve head astrocytes were positive for the astrocyte marker GFAP and negative for markers for microglia (F4/80) and oligodendrocytes (OSP1). Significantly increased %CLAN-positive cells were observed after 48-h treatment with TGFß2 vs. control in a dose-dependent manner. Co-treatment with GsMTx4 significantly decreased %CLAN-positive cells vs. TGFß2 treatment and the presence of f-actin stress fibers. TGFß2 treatment significantly increased GFAP, fibronectin, and collagen IV levels, and GsMTx4 co-treatment ameliorated GFAP immunoreactivity. Our data suggest inhibition of mechanosensitive channel activity as a potential therapeutic strategy to modulate actin cytoskeletal remodeling within the optic nerve head in glaucoma.
Assuntos
Actinas/metabolismo , Astrócitos/metabolismo , Citoesqueleto/metabolismo , Glaucoma/metabolismo , Pressão Intraocular/fisiologia , Disco Óptico/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Animais , Astrócitos/patologia , Células Cultivadas , Citoesqueleto/patologia , Modelos Animais de Doenças , Glaucoma/patologia , Glaucoma/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Disco Óptico/patologiaRESUMO
Many studies have demonstrated that rhegmatogenous retinal detachment (RRD) leads to impaired retinal circulation. However, the involvement of inflammation in the RRD-induced worsening of retinal circulation was obscure. This retrospective observational study included 150 patients with primary RRD (macula-on, n = 63; macula-off, n = 87) who underwent 25-gauge microincision vitrectomy surgery (25G MIVS). Total retinal blood flow was represented by the mean blur rate (MBR) of the optic nerve head vessel, measured by laser speckle flowgraphy preoperatively and until 6 months postoperatively. Aqueous humor samples were obtained during surgery to determine cytokine concentrations by enzyme-linked immunosorbent assay. At 3 and 6 months postoperatively, there were no significant differences between eyes with macula-on RRD and fellow eyes. However, in macula-off RRD, MBR remained significantly lower in RRD eyes 6 months postoperatively (P < 0.05). Log-transformed levels of soluble intercellular adhesion molecule-1 (sICAM-1) were negatively correlated with relative MBR (r-MBR, RRD eye/fellow eye) before surgery (r = - 0.47, P = 0.01) in macula-on, but not macula-off, RRD. Six months postoperatively, r-MBR correlated significantly with sICAM-1 levels (r = - 0.36, P = 0.02) in macula-off RRD. ICAM-1 may play a role in RRD-induced deterioration of retinal circulation.
Assuntos
Oftalmopatias Hereditárias/genética , Molécula 1 de Adesão Intercelular/genética , Macula Lutea/metabolismo , Retina/metabolismo , Descolamento Retiniano/genética , Oftalmopatias Hereditárias/sangue , Oftalmopatias Hereditárias/patologia , Oftalmopatias Hereditárias/cirurgia , Feminino , Humanos , Molécula 1 de Adesão Intercelular/sangue , Macula Lutea/irrigação sanguínea , Macula Lutea/patologia , Macula Lutea/cirurgia , Masculino , Pessoa de Meia-Idade , Disco Óptico/metabolismo , Disco Óptico/patologia , Retina/patologia , Retina/cirurgia , Descolamento Retiniano/sangue , Descolamento Retiniano/patologia , Descolamento Retiniano/cirurgia , Tomografia de Coerência Óptica , Acuidade Visual/genética , Acuidade Visual/fisiologia , VitrectomiaRESUMO
Glaucoma is a group of optic neuropathies that leads to irreversible vision loss. The optic nerve head (ONH) is the site of initial optic nerve damage in glaucoma. ONH-derived lamina cribrosa (LC) cells synthesize extracellular matrix (ECM) proteins; however, these cells are adversely affected in glaucoma and cause detrimental changes to the ONH. LC cells respond to mechanical strain by increasing the profibrotic cytokine transforming growth factor-beta 2 (TGFß2) and ECM proteins. Moreover, microRNAs (miRNAs or miR) regulate ECM gene expression in different fibrotic diseases, including glaucoma. A delicate homeostatic balance between profibrotic and anti-fibrotic miRNAs may contribute to the remodeling of ONH. This study aimed to determine whether modulation of miRNAs alters the expression of ECM in human LC cells. Primary human normal and glaucoma LC cells were grown to confluency and treated with or without TGFß2 for 24 h. Differences in expression of miRNAs were analyzed using miRNA qPCR arrays. miRNA PCR arrays showed that the miR-29 family was significantly decreased in glaucomatous LC cell strains compared to age-matched controls. TGFß2 treatment downregulated the expression of multiple miRNAs, including miR-29c-3p, compared to controls in LC cells. LC cells transfected with miR-29c-3p mimics or inhibitors modulated collagen expression.
Assuntos
Regulação da Expressão Gênica , Glaucoma/genética , MicroRNAs/genética , Disco Óptico/metabolismo , Doenças do Nervo Óptico/genética , Fator de Crescimento Transformador beta2/farmacologia , Estudos de Casos e Controles , Perfilação da Expressão Gênica , Glaucoma/tratamento farmacológico , Glaucoma/patologia , Humanos , Disco Óptico/efeitos dos fármacos , Disco Óptico/patologia , Doenças do Nervo Óptico/tratamento farmacológico , Doenças do Nervo Óptico/patologiaRESUMO
Purpose: The lamina cribrosa (LC) is a key site of damage in glaucomatous optic neuropathy. We previously found that glaucoma LC cells have an increased profibrotic gene expression, with mitochondrial dysfunction in the form of decreased mitochondrial membrane potential. Altered cell bioenergetics have recently been reported in organ fibrosis and in cancer. In this study, we carried out a systematic mitochondrial bioenergetic assessment and measured markers of alternative sources of cellular energy in normal and glaucoma LC cells. Methods: LC cells from three glaucoma donors and three age-matched normal controls were assessed using VICTOR X4 Perkin Elmer (Waltham, MA) plate reader with different phosphorescent and luminescent probes. adenosine triphosphate levels, oxygen consumption rate, and extracellular acidification were measured and normalized to total protein content. RNA and protein expression levels of MCT1, MCT4, MTFHD2, and GLS2 were quantified using real-time RT-PCR and Western blotting. Results: Glaucoma LC cells contain significantly less adenosine triphosphate (P < .05) when supplied with either glucose or galactose. They also showed significantly diminished oxygen consumption in both basal and maximal respiration with more lactic acid contribution in ECA. Both mRNA and protein expression levels of MCT1, MCT4, MTHFD2, and GLS2 were significantly increased in glaucoma LC cells. Conclusions: We demonstrate evidence of metabolic reprogramming (The Warburg effect) in glaucoma LC cells. Expression of markers of glycolysis, glutamine, and one carbon metabolism are elevated in glaucoma cells at both the mRNA and protein levels. A better understanding of bioenergetics in glaucoma may help in the development of new therapeutics.
Assuntos
Glaucoma de Ângulo Aberto/metabolismo , Glicólise/fisiologia , Doenças Mitocondriais/metabolismo , Disco Óptico/metabolismo , Doenças do Nervo Óptico/metabolismo , Fosforilação Oxidativa , Trifosfato de Adenosina/metabolismo , Aminoidrolases/genética , Aminoidrolases/metabolismo , Biomarcadores , Western Blotting , Células Cultivadas , Perfilação da Expressão Gênica , Glaucoma de Ângulo Aberto/patologia , Proteína Glial Fibrilar Ácida/metabolismo , Glutaminase/genética , Glutaminase/metabolismo , Humanos , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Doenças Mitocondriais/patologia , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Disco Óptico/patologia , Doenças do Nervo Óptico/patologia , Consumo de Oxigênio/fisiologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Simportadores/genética , Simportadores/metabolismo , Doadores de TecidosRESUMO
BACKGROUND: Alzheimer's disease (AD) pathology precedes symptoms and its detection can identify at-risk individuals who may benefit from early treatment. Since the retinal nerve fiber layer (RNFL) is depleted in established AD, we tested whether its thickness can predict whether cognitively healthy (CH) individuals have a normal or pathological cerebrospinal fluid (CSF) Aß42 (A) and tau (T) ratio. METHODS: As part of an ongoing longitudinal study, we enrolled CH individuals, excluding those with cognitive impairment and significant ocular pathology. We classified the CH group into two sub-groups, normal (CH-NAT, n = 16) or pathological (CH-PAT, n = 27), using a logistic regression model from the CSF AT ratio that identified >85% of patients with a clinically probable AD diagnosis. Spectral-domain optical coherence tomography (OCT) was acquired for RNFL, ganglion cell-inner plexiform layer (GC-IPL), and macular thickness. Group differences were tested using mixed model repeated measures and a classification model derived using multiple logistic regression. RESULTS: Mean age (± standard deviation) in the CH-PAT group (n = 27; 75.2 ± 8.4 years) was similar (p = 0.50) to the CH-NAT group (n = 16; 74.1 ± 7.9 years). Mean RNFL (standard error) was thinner in the CH-PAT group by 9.8 (2.7) µm; p < 0.001. RNFL thickness classified CH-NAT vs. CH-PAT with 87% sensitivity and 56.3% specificity. CONCLUSIONS: Our retinal data predict which individuals have CSF biomarkers of AD pathology before cognitive deficits are detectable with 87% sensitivity. Such results from easy-to-acquire, objective and non-invasive measurements of the RNFL merit further study of OCT technology to monitor or screen for early AD pathology.
Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Disfunção Cognitiva/genética , Proteínas tau/genética , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Amiloidose/líquido cefalorraquidiano , Amiloidose/diagnóstico por imagem , Amiloidose/genética , Amiloidose/patologia , Biomarcadores/líquido cefalorraquidiano , Disfunção Cognitiva/líquido cefalorraquidiano , Disfunção Cognitiva/diagnóstico por imagem , Disfunção Cognitiva/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fibras Nervosas/metabolismo , Fibras Nervosas/patologia , Disco Óptico/diagnóstico por imagem , Disco Óptico/metabolismo , Disco Óptico/patologia , Retina/diagnóstico por imagem , Retina/metabolismo , Retina/patologia , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Tomografia de Coerência Óptica , Proteínas tau/líquido cefalorraquidianoRESUMO
Glaucoma is the leading cause of irreversible vision loss. Ocular hypertension is a major risk factor for glaucoma and recent work has demonstrated critical early neuroinflammatory insults occur in the optic nerve head following ocular hypertension. Microglia and infiltrating monocytes are likely candidates to drive these neuroinflammatory insults. However, the exact molecular identity / transcriptomic profile of microglia following ocular hypertensive insults is unknown. To elucidate the molecular identity of microglia after long-term exposure to ocular hypertension, we used a mouse model of glaucoma (DBA/2 J). We performed RNA-sequencing of microglia mRNA from the optic nerve head at a time point following ocular hypertensive insults, but preceding detectable neurodegeneration (with microglia identified as being CD45lo/CD11b+/CD11c-). Furthermore, RNA-sequencing was performed on optic nerve head microglia from mice treated with radiation therapy, a potent therapy preventing neuroinflammatory insults. Transcriptomic profiling of optic nerve head microglia mRNA identifies metabolic priming with marked changes in mitochondrial gene expression, and changes to phagocytosis, inflammatory, and sensome pathways. The data predict that many functions of microglia that help maintain tissue homeostasis are affected. Comparative analysis of these data with data from previously published whole optic nerve head tissue or monocyte-only samples from DBA/2 J mice demonstrate that many of the neuroinflammatory signatures in these data sets arise from infiltrating monocytes and not reactive microglia. Finally, our data demonstrate that prophylactic radiation therapy of DBA/2 J mice potently abolishes these microglia metabolic transcriptomic changes at the same time points. Together, our data provide a unique resource for the community to help drive further hypothesis generation and testing in glaucoma.
Assuntos
Regulação da Expressão Gênica , Homeostase/genética , Microglia/metabolismo , Microglia/patologia , Hipertensão Ocular/genética , Hipertensão Ocular/patologia , Disco Óptico/metabolismo , Animais , Regulação para Baixo/genética , Feminino , Regulação da Expressão Gênica/efeitos da radiação , Homeostase/efeitos da radiação , Pressão Intraocular/genética , Pressão Intraocular/efeitos da radiação , Camundongos Endogâmicos DBA , Microglia/efeitos da radiação , Monócitos/metabolismo , Monócitos/patologia , Hipertensão Ocular/fisiopatologia , Disco Óptico/patologia , Disco Óptico/efeitos da radiação , Fagocitose/genética , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/genética , Regulação para Cima/genéticaRESUMO
Purpose: To investigate the function and expression of the PGE2 receptors EP1-4 in rat retinal ischemia-reperfusion (I/R) injury and to determine the regulatory role of resveratrol (RES) in this process. Methods: In vitro, we stimulated primary astrocytes extracted from the optic disc of rats with epidermal growth factor (EGF) and RES, and detected the location of EP1-4 expression with immunofluorescence. The expression of antiglial fibrillary acidic protein (GFAP), EGF receptor (EGFR), inducible NOS (iNOS), and EP1-4 in astrocytes was detected with western blotting. In vivo, we established an I/R injury model and RES treatment model with Sprague-Dawley rats. Changes in the thickness of the inner retina were observed with hematoxylin and eosin (H&E) staining. EP1-4 localization in the retina was observed with immunohistochemistry. The expression of COX-2, iNOS, and EP1-4 in the control and model groups was detected with western blotting. Results: In this study, immunofluorescence and immunohistochemistry showed that EP1-4 are expressed in astrocytes and the rat retina. EGF stimulation increased the expression of EGFR, iNOS, EP1, EP2, and EP4 in astrocytes. The expression of EP1-4 was statistically significantly increased on the third day after model induction, and EP1-4 expression decreased to normal levels on day 7. EGF and RES mediated the decrease in the expression of EP2. RES treatment significantly reduced retinal damage and RGC loss, as demonstrated by the relatively intact tissue structure on day 7 observed with H&E staining. Moreover, inflammation was associated with this I/R injury model, as demonstrated by the early induction of proinflammatory mediators, and this inflammation was significantly attenuated after RES treatment. Conclusions: These results indicate that the COX-2/PGE2/EPs pathway is involved in retinal damage and astrocyte inflammation. In addition, the results suggest that the neuroprotective effects of RES may be associated with decreased production of inflammatory mediators. These results suggest that the PGE2 receptor may be a key factor in the treatment of neurodegenerative diseases, and that RES may be used as a possible therapeutic strategy for glaucoma.
Assuntos
Astrócitos/metabolismo , Disco Óptico/metabolismo , Receptores de Prostaglandina E/metabolismo , Traumatismo por Reperfusão/metabolismo , Retina/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Modelos Animais de Doenças , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Imuno-Histoquímica , Inflamação/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/metabolismo , Disco Óptico/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/tratamento farmacológico , Resveratrol/farmacologia , Retina/efeitos dos fármacos , Retina/patologia , Transdução de Sinais/genéticaRESUMO
Optic nerve head morphology is affected by several retinal diseases. We measured the vertical optic disc diameter (DD) of the UK Biobank (UKBB) cohort (N = 67 040) and performed the largest genome-wide association study (GWAS) of DD to date. We identified 81 loci (66 novel) for vertical DD. We then replicated the novel loci in International Glaucoma Genetic Consortium (IGGC, N = 22 504) and European Prospective Investigation into Cancer-Norfolk (N = 6005); in general the concordance in effect sizes was very high (correlation in effect size estimates 0.90): 44 of the 66 novel loci were significant at P < 0.05, with 19 remaining significant after Bonferroni correction. We identified another 26 novel loci in the meta-analysis of UKBB and IGGC data. Gene-based analyses identified an additional 57 genes. Human ocular tissue gene expression analysis showed that most of the identified genes are enriched in optic nerve head tissue. Some of the identified loci exhibited pleiotropic effects with vertical cup-to-disc ratio, intraocular pressure, glaucoma and myopia. These results can enhance our understanding of the genetics of optic disc morphology and shed light on the genetic findings for other ophthalmic disorders such as glaucoma and other optic nerve diseases.
Assuntos
Estudo de Associação Genômica Ampla , Glaucoma/genética , Disco Óptico/anatomia & histologia , Adulto , Idoso , Bases de Dados Factuais , Feminino , Expressão Gênica , Glaucoma/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Disco Óptico/metabolismo , Polimorfismo de Nucleotídeo Único , Estudos ProspectivosRESUMO
Purpose: We assess if α retinal ganglion cells (αRGCs) and intrinsically photosensitive retinal ganglion cells (ipRGCs) survive in mouse models of glaucoma. Methods: Two microliters of N-methyl-D-aspartate (NMDA; 1 mM) or PBS were injected intraocularly 7 days before sacrifice. Immunohistochemical analyses of the retina were performed using antibodies against RNA-binding protein with multiple splicing (RBPMS), osteopontin, and melanopsin. Immunohistochemical analyses also were performed in adult mice with glutamate/aspartate transporter (GLAST) deletion (GLAST knockout [KO] mice), a mouse model of normal tension glaucoma. Results: NMDA-induced loss of RBPMS-positive total RGCs was 58.4% ± 0.4% compared to PBS-treated controls, whereas the loss of osteopontin-positive αRGCs was 5.0% ± 0.6% and that of melanopsin-positive ipRGCs was 7.6% ± 1.6%. In GLAST KO mice, the loss of total RGCs was 48.4% ± 0.9% compared to wild-type mice, whereas the loss of αRGCs and ipRGCs was 3.9% ± 0.4% and 9.3% ± 0.5%, respectively. The distribution of survived total RGCs, αRGCs, and ipRGCs was similar regardless of the location of the retina. Conclusions: These results suggest that αRGC and ipRGC are highly tolerant to NMDA-induced neurotoxicity and NTG-like neurodegeneration in GLAST KO mice.
Assuntos
Modelos Animais de Doenças , Agonistas de Aminoácidos Excitatórios/toxicidade , Glaucoma de Baixa Tensão/prevenção & controle , N-Metilaspartato/toxicidade , Disco Óptico/efeitos dos fármacos , Células Ganglionares da Retina/efeitos dos fármacos , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Sobrevivência Celular/fisiologia , Técnica Indireta de Fluorescência para Anticorpo , Injeções Intravítreas , Luz , Glaucoma de Baixa Tensão/metabolismo , Glaucoma de Baixa Tensão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Disco Óptico/metabolismo , Disco Óptico/efeitos da radiação , Osteopontina/metabolismo , Proteínas de Ligação a RNA/metabolismo , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Degeneração Retiniana/prevenção & controle , Células Ganglionares da Retina/classificação , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/efeitos da radiaçãoRESUMO
Purpose: The purpose of this study was to measure the 2D collagen network structure of the human lamina cribrosa (LC), analyze for the correlations with age, region, and LC size, as well as the correlations with pressure-induced strains. Methods: The posterior scleral cups of 10 enucleated human eyes with no known ocular disease were subjected to ex vivo inflation testing from 5 to 45 mm Hg. The optic nerve head was imaged by using second harmonic generation imaging (SHG) to identify the LC collagen structure at both pressures. Displacements and strains were calculated by using digital volume correlation of the SHG volumes. Nine structural features were measured by using a custom Matlab image analysis program, including the pore area fraction, node density, and beam connectivity, tortuosity, and anisotropy. Results: All strain measures increased significantly with higher pore area fraction, and all but the radial-circumferential shear strain (Erθ) decreased with higher node density. The maximum principal strain (Emax) and maximum shear strain (Γmax) also increased with larger beam aspect ratio and tortuosity, respectively, and decreased with higher connectivity. The peripheral regions had lower node density and connectivity, and higher pore area fraction, tortuosity, and strains (except for Erθ) than the central regions. The peripheral nasal region had the lowest Emax, Γmax, radial strain, and pore area fraction. Conclusions: Features of LC beam network microstructure that are indicative of greater collagen density and connectivity are associated with lower pressure-induced LC strain, potentially contributing to resistance to glaucomatous damage.
Assuntos
Módulo de Elasticidade/fisiologia , Colágenos Fibrilares/metabolismo , Pressão Intraocular/fisiologia , Disco Óptico/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/fisiologia , Anisotropia , Fenômenos Biomecânicos , Enucleação Ocular , Feminino , Humanos , Imageamento Tridimensional , Masculino , Pessoa de Meia-Idade , Disco Óptico/diagnóstico por imagem , Estresse Mecânico , Doadores de TecidosRESUMO
Glaucoma is a leading cause of irreversible vision loss and is associated with fibrotic changes in two ocular tissues-the optic nerve head (ONH) and trabecular meshwork (TM). We investigated the differences in extracellular matrix components (ECM) including collagen, elastin, transforming growth factor beta-2, type-II receptor (TGFßRII) and Galectin3 (Gal3) in the glaucomatous human eyes to quantify fibrotic changes in ONH and TM. Glaucomatous and control human donor eyes were prepared for chemical and immunological staining to quantify ECM protein expression in the TM and ONH. Chemical staining included: Trichrome (collagen), Vernhoeff-Van Giesen (elastin) and Sirius Red (collagen). Immunohistochemistry was used to determine levels of Gal3 and TGFß2RII. Quantitative analyses were performed using Image J software. Student's t-test was used to compare groups and Pearson's test was used to determine correlations P-values of 0.05 (or less) were considered statistically significant. Deposition of ECM proteins was elevated in glaucomatous tissues. There was increased collagen (P = 0.0469), Gal3 (P < 0.0001) and TGFß2RII (P = 0.0005) in the TM of glaucomatous eyes. Likewise, collagen (P = 0.0517) and Galectin3 (P = 0.041) were increased in the ONH glaucomatous eyes. There was a correlation of TGFßRII with Gal3 in the TM (P < 0.0001) and optic nerve (P = 0.0003). The TM and ONH of glaucomatous eyes showed increased expression of ECM proteins supporting a fibrotic pathology. Galectin3 and TGFß-2R II showed a positive correlation in TM and optic nerve supporting co-localization and suggesting their potential role in the glaucoma fibrotic process. Clin. Anat. 31:1031-1049, 2018. © 2018 The Authors. Clinical Anatomy published by Wiley Periodicals, Inc. on behalf of American Association of Clinical Anatomists.
Assuntos
Galectina 3/metabolismo , Glaucoma/metabolismo , Disco Óptico/metabolismo , Malha Trabecular/metabolismo , Idoso , Idoso de 80 Anos ou mais , Proteínas Sanguíneas , Estudos de Casos e Controles , Proteínas da Matriz Extracelular/metabolismo , Colágenos Fibrilares/metabolismo , Fibrose , Galectinas , Glaucoma/patologia , Humanos , Disco Óptico/patologia , Malha Trabecular/patologia , Fator de Crescimento Transformador beta2/metabolismoRESUMO
Statins are cholesterol lowering drugs and have shown beneficial effects on glaucoma. With regard to the mechanism of statin action on glaucoma, we investigated the effects of statins on transforming growth factor-beta 2 (TGF-ß2)-induced expression of extracellular matrix (ECM) proteins in human astrocytes of the optic nerve head (ONH) lamina cribrosa (LC). By using primary human ONH astrocytes, we found that both simvastatin and lovastatin inhibited TGF-ß2-mediated expression of ECM proteins such as connective tissue growth factor, collagen I, fibronectin, and plasminogen activator inhibitor-1. Suppression of ECM related proteins is due to inhibition of Smad2/3 activation as statins inhibit TGF-ß2-induced Smad2 phosphorylation and Smad2/3 nuclear accumulation. In ONH astrocytes, TGF-ß2 does not induce MAPK activation. In this study we found an anti-fibrotic effect of statins in human astrocytes of the ONH and identified TGF-ß2 as a mediator of statin action, which may support a beneficial role for statins in blocking glaucomatous axonal damage induced by ECM remodeling.
Assuntos
Astrócitos/efeitos dos fármacos , Matriz Extracelular/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Disco Óptico/metabolismo , Fator de Crescimento Transformador beta2/fisiologia , Análise de Variância , Astrócitos/metabolismo , Células Cultivadas , Matriz Extracelular/efeitos dos fármacos , Proteínas do Olho/metabolismo , Humanos , Lovastatina , Disco Óptico/citologia , Sinvastatina , Fator de Crescimento Transformador beta2/metabolismoRESUMO
BACKGROUND: We previously reported a profound long-term neuroprotection subsequent to a single radiation-therapy in the DBA/2J mouse model of glaucoma. This neuroprotection prevents entry of monocyte-like immune cells into the optic nerve head during glaucoma. Gene expression studies in radiation-treated mice implicated Glycam1 in this protection. Glycam1 encodes a proteoglycan ligand for L-selectin and is an excellent candidate to modulate immune cell entry into the eye. Here, we experimentally test the hypothesis that radiation-induced over-expression of Glycam1 is a key component of the neuroprotection. METHODS: We generated a null allele of Glycam1 on a DBA/2J background. Gene and protein expression of Glycam1, monocyte entry into the optic nerve head, retinal ganglion cell death, and axon loss in the optic nerve were assessed. RESULTS: Radiation therapy potently inhibits monocyte entry into the optic nerve head and prevents retinal ganglion cell death and axon loss. DBA/2J mice carrying a null allele of Glycam1 show increased monocyte entry and increased retinal ganglion cell death and axon loss following radiation therapy, but the majority of optic nerves were still protected by radiation therapy. CONCLUSIONS: Although GlyCAM1 is an L-selectin ligand, its roles in immunity are not yet fully defined. The current study demonstrates a partial role for GlyCAM1 in radiation-mediated protection. Furthermore, our results clearly show that GlyCAM1 levels modulate immune cell entry from the vasculature into neural tissues. As Glycam1 deficiency has a more profound effect on cell entry than on neurodegeneration, further experiments are needed to precisely define the role of monocyte entry in DBA/2J glaucoma. Nevertheless, GlyCAM1's function as a negative regulator of extravasation may lead to novel therapeutic strategies for an array of common conditions involving inflammation.
Assuntos
Glaucoma/metabolismo , Glaucoma/radioterapia , Monócitos/metabolismo , Mucinas/biossíntese , Mucinas/efeitos da radiação , Disco Óptico/metabolismo , Animais , Feminino , Glaucoma/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Disco Óptico/irrigação sanguínea , Nervo Óptico/irrigação sanguínea , Nervo Óptico/metabolismoAssuntos
Neuropatias Amiloides Familiares/tratamento farmacológico , Benzoxazóis/uso terapêutico , Iris/patologia , Disco Óptico/patologia , Epitélio Pigmentado da Retina/patologia , Corpo Vítreo/patologia , Amiloide/química , Amiloide/metabolismo , Neuropatias Amiloides Familiares/metabolismo , Neuropatias Amiloides Familiares/patologia , Progressão da Doença , Feminino , Humanos , Iris/efeitos dos fármacos , Iris/metabolismo , Masculino , Pessoa de Meia-Idade , Disco Óptico/efeitos dos fármacos , Disco Óptico/metabolismo , Pré-Albumina/química , Pré-Albumina/metabolismo , Agregados Proteicos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Corpo Vítreo/efeitos dos fármacos , Corpo Vítreo/metabolismoRESUMO
The induction and patterning of the conjunctival papillae (i.e. epithelial thickenings of the conjunctiva required for the induction of the underlying, neural crest-derived scleral ossicles) is complex. It takes place over a period of two days and follows a defined spatiotemporal sequence. In this study, we investigated the spatial and temporal expression pattern of four genes over seven morphological stages of development of these papillae. We show that ß-catenin is expressed during the pre-patterning of the epithelium prior to papilla induction and second that ß-catenin, Ednrb and Inhba are expressed during the induction and patterning of the conjunctival papillae. Furthermore, we identified two genes, ß-catenin and Prox1, that may be involved in the induction of the underlying scleral bones. These data provide an excellent baseline for future studies, setting the stage for functional studies aimed at examining the role of these genes in the patterning of the scleral ossicle system. This study also outlines the similarities between the conjunctival papillae and other placodes and may provide insights into the evolution and development of the conjunctival papillae.
Assuntos
Embrião de Galinha/crescimento & desenvolvimento , Olho/metabolismo , Subunidades beta de Inibinas/genética , Receptor de Endotelina B/genética , beta Catenina/genética , Animais , Padronização Corporal/genética , Embrião de Galinha/metabolismo , Túnica Conjuntiva/crescimento & desenvolvimento , Túnica Conjuntiva/metabolismo , Desenvolvimento Embrionário/genética , Epitélio/crescimento & desenvolvimento , Epitélio/metabolismo , Olho/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Subunidades beta de Inibinas/biossíntese , Crista Neural/crescimento & desenvolvimento , Crista Neural/metabolismo , Disco Óptico/crescimento & desenvolvimento , Disco Óptico/metabolismo , Receptor de Endotelina B/biossíntese , Transdução de Sinais/genética , beta Catenina/biossínteseRESUMO
Cell adhesion molecules play a central role in mediating axonal tract development within the nascent nervous system. NF-protocadherin (NFPC), a member of the non-clustered protocadherin family, has been shown to regulate retinal ganglion cell (RGC) axon and dendrite initiation, as well as influencing axonal navigation within the mid-optic tract. However, whether NFPC mediates RGC axonal behaviour at other positions within the optic pathway remains unclear. Here we report that NFPC plays an important role in RGC axonogenesis, but not in intraretinal guidance. Moreover, axons with reduced NFPC levels exhibit insensitivity to Netrin-1, an attractive guidance cue expressed at the optic nerve head. Netrin-1 induces rapid turnover of NFPC localized to RGC growth cones, suggesting that the regulation of NFPC protein levels may underlie Netrin-1-mediated entry of RGC axons into the optic nerve head. At the tectum, we further reveal a function for NFPC in controlling RGC axonal entry into the final target area. Collectively, our results expand our understanding of the role of NFPC in RGC guidance and illustrate that this adhesion molecule contributes to axon behaviour at multiple points in the optic pathway.
Assuntos
Axônios/metabolismo , Caderinas/metabolismo , Células Ganglionares da Retina/metabolismo , Vias Visuais/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Moléculas de Adesão Celular/metabolismo , Dendritos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Fatores de Crescimento Neural/metabolismo , Netrina-1 , Neurogênese/fisiologia , Disco Óptico/metabolismo , Protocaderinas , Retina , Proteínas Supressoras de Tumor/metabolismo , Xenopus laevis/metabolismo , Xenopus laevis/fisiologiaRESUMO
We report on clinical, genetic and metabolic investigations in a family with optic neuropathy, non-progressive cardiomyopathy and cognitive disability. Ophthalmic investigations (slit lamp examination, funduscopy, OCT scan of the optic nerve, ERG and VEP) disclosed mild or no decreased visual acuity, but pale optic disc, loss of temporal optic fibers and decreased VEPs. Mitochondrial DNA and exome sequencing revealed a novel homozygous mutation in the nuclear MTO1 gene and the homoplasmic m.593T>G mutation in the mitochondrial MT-TF gene. Muscle biopsy analyses revealed decreased oxygraphic Vmax values for complexes I+III+IV, and severely decreased activities of the respiratory chain complexes (RCC) I, III and IV, while muscle histopathology was normal. Fibroblast analysis revealed decreased complex I and IV activity and assembly, while cybrid analysis revealed a partial complex I deficiency with normal assembly of the RCC. Thus, in patients with a moderate clinical presentation due to MTO1 mutations, the presence of an optic atrophy should be considered. The association with the mitochondrial mutation m.593T>G could act synergistically to worsen the complex I deficiency and modulate the MTO1-related disease.
Assuntos
Cardiomiopatias/genética , Proteínas de Transporte/genética , Homozigoto , Deficiência Intelectual/genética , Mutação , Doenças do Nervo Óptico/genética , RNA de Transferência de Fenilalanina/genética , Adulto , Cardiomiopatias/complicações , Cardiomiopatias/diagnóstico , Cardiomiopatias/patologia , Análise Mutacional de DNA , Complexo I de Transporte de Elétrons/genética , Complexo II de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Expressão Gênica , Humanos , Deficiência Intelectual/complicações , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/patologia , Masculino , Potencial da Membrana Mitocondrial/genética , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Disco Óptico/metabolismo , Disco Óptico/patologia , Doenças do Nervo Óptico/complicações , Doenças do Nervo Óptico/diagnóstico , Doenças do Nervo Óptico/patologia , Linhagem , Proteínas de Ligação a RNA , Acuidade VisualRESUMO
PURPOSE: To review the current literature regarding the role of matricellular proteins in glaucoma, specifically in the lamina cribrosa (LC) region of the optic nerve head (ONH) and the trabecular meshwork (TM). METHODS: A literature search was performed for published articles describing the expression and function of matricellular proteins such as thrombospondin (TSP), connective tissue growth factor (CTGF), secreted protein acidic and rich in cysteine (SPARC), and periostin in glaucoma. RESULTS: In glaucoma, there are characteristic extracellular matrix (ECM) changes associated with optic disc cupping in the ONH and subsequent visual field defects. Matricellular proteins are a family of nonstructural secreted glycoproteins, which enable cells to communicate with their surrounding ECM, including CTGF, also known as CCN2, TSPs, SPARC, periostin, osteonectin, and tenascin-C and -X, and other ECM proteins. Such proteins appear to play a role in fibrosis and increased ECM deposition. Importantly, most are widely expressed in tissues particularly in the TM and ONH, and deficiency of TSP1 and SPARC has been shown to lower intraocular pressure in mouse models of glaucoma through enhanced outflow facility. CONCLUSION: This article highlights the role of matricellular proteins in glaucoma pathology. The potential role of these proteins in glaucoma is emerging as some have an association with the pathophysiology of the TM and LC region and might therefore be potential targets for therapeutic intervention in glaucoma.