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Purpose: Accumulation of lysosomal waste is linked to neurodegeneration in multiple diseases, and pharmacologic enhancement of lysosomal activity is hypothesized to reduce pathology. An excessive accumulation of lysosomal-associated lipofuscin waste and an elevated lysosomal pH occur in retinal pigment epithelial cells of the ABCA4-/- mouse model of Stargardt's retinal degeneration. As treatment with the P2Y12 receptor antagonist ticagrelor was previously shown to lower lysosomal pH and lipofuscin-like autofluorescence in these cells, we asked whether oral delivery of ticagrelor also prevented photoreceptor loss. Methods: Moderate light exposure was used to accelerate photoreceptor loss in albino ABCA4-/- mice as compared to BALB/c controls. Ticagrelor (0.1%-0.15%) was added to mouse chow for between 1 and 10 months. Photoreceptor function was determined with electroretinograms, while cell survival was determined using optical coherence tomography and histology. Results: Protection by ticagrelor was demonstrated functionally by using the electroretinogram, as ticagrelor-treated ABCA4-/- mice had increased a- and b-waves compared to untreated mice. Mice receiving ticagrelor treatment had a thicker outer nuclear layer, as measured with both optical coherence tomography and histologic sections. Ticagrelor decreased expression of LAMP1, implicating enhanced lysosomal function. No signs of retinal bleeding were observed after prolonged treatment with ticagrelor. Conclusions: Oral treatment with ticagrelor protected photoreceptors in the ABCA4-/- mouse, which is consistent with enhanced lysosomal function. As mouse ticagrelor exposure levels were clinically relevant, the drug may be of benefit in preventing the loss of photoreceptors in Stargardt's disease and other neurodegenerations associated with lysosomal dysfunction.
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Degeneração Retiniana/prevenção & controle , Epitélio Pigmentado da Retina/patologia , Ticagrelor/administração & dosagem , Administração Oral , Animais , Modelos Animais de Doenças , Eletrorretinografia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana Lisossomal/biossíntese , Proteínas de Membrana Lisossomal/genética , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias , Antagonistas do Receptor Purinérgico P2Y/administração & dosagem , RNA/genética , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/fisiopatologia , Tomografia de Coerência Óptica , Resultado do TratamentoRESUMO
The accumulation of partially degraded lipid waste in lysosomal-related organelles may contribute to pathology in many aging diseases. The presence of these lipofuscin granules is particularly evident in the autofluorescent lysosome-associated organelles of the retinal pigmented epithelial (RPE) cells, and may be related to early stages of age-related macular degeneration. While lysosomal enzymes degrade material optimally at acidic pH levels, lysosomal pH is elevated in RPE cells from the ABCA4-/- mouse model of Stargardt's disease, an early onset retinal degeneration. Lowering lysosomal pH through cAMP-dependent pathways decreases accumulation of autofluorescent material in RPE cells in vitro, but identification of an appropriate receptor is crucial for manipulating this pathway in vivo. As the P2Y12 receptor for ADP is coupled to the inhibitory Gi protein, we asked whether blocking the P2Y12 receptor with ticagrelor could restore lysosomal acidity and reduce autofluorescence in compromised RPE cells from ABCA4-/- mice. Oral delivery of ticagrelor giving rise to clinically relevant exposure lowered lysosomal pH in these RPE cells. Ticagrelor also partially reduced autofluorescence in the RPE cells of ABCA4-/- mice. In vitro studies in ARPE-19 cells using more specific antagonists AR-C69931 and AR-C66096 confirmed the importance of the P2Y12 receptor for lowering lysosomal pH and reducing autofluorescence. These observations identify P2Y12 receptor blockade as a potential target to lower lysosomal pH and clear lysosomal waste in RPE cells.
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Mechanical strain in neural tissues can lead to the up-regulation and release of multiple cytokines including interleukin 6 (IL-6). In the retina, the mechanosensitive release of ATP can autostimulate P2X7 receptors on both retinal ganglion cell neurons and optic nerve head astrocytes. Here, we asked whether the purinergic signaling contributed to the IL-6 response to increased intraocular pressure (IOP) in vivo, and stretch or swelling in vitro. Rat and mouse eyes were exposed to non-ischemic elevations in IOP to 50-60 mmHg for 4 h. A PCR array was used to screen cytokine changes, with quantitative (q)PCR used to confirm mRNA elevations and immunoblots used for protein levels. P2X7 antagonist Brilliant Blue G (BBG) and agonist (4-benzoyl-benzoyl)-ATP (BzATP) were injected intravitreally. ELISA was used to quantify IL-6 release from optic nerve head astrocytes or retinal ganglion cells. Receptor identity was confirmed pharmacologically and in P2X7-/- mice, acute elevation of IOP altered retinal expression of multiple cytokine genes. Elevation of IL-6 was greatest, with expression of IL1rn, IL24, Tnf, Csf1, and Lif also increased more than twofold, while expression of Tnfsf11, Gdf9, and Tnfsf4 were reduced. qPCR confirmed the rise in IL-6 and extracellular ATP marker ENTPD1, but not pro-apoptotic genes. Intravitreal injection of P2X7 receptor antagonist BBG prevented the pressure-dependent rise in IL-6 mRNA and protein in the rat retina, while injection of P2X7 receptor agonist BzATP was sufficient to elevate IL-6 expression. IOP elevation increased IL-6 in wild-type but not P2X7R knockout mice. Application of mechanical strain to isolated optic nerve head astrocytes increased IL-6 levels. This response was mimicked by agonist BzATP, but blocked by antagonists BBG and A839977. Stretch or BzATP led to IL-6 release from both astrocytes and isolated retinal ganglion cells. The mechanosensitive up-regulation and release of cytokine IL-6 from the retina involves the P2X7 receptor, with both astrocytes and neurons contributing to the response.
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Astrócitos/metabolismo , Interleucina-6/fisiologia , Neurônios/metabolismo , Receptores Purinérgicos P2X7/fisiologia , Trifosfato de Adenosina/administração & dosagem , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Injeções , Interleucina-6/genética , Pressão Intraocular , Camundongos , Camundongos Knockout , Nervo Óptico/patologia , Agonistas do Receptor Purinérgico P2X/administração & dosagem , Agonistas do Receptor Purinérgico P2X/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Ratos , Ratos Long-Evans , Ratos Sprague-Dawley , Receptores Purinérgicos P2X7/genética , Células Ganglionares da Retina/efeitos dos fármacos , Regulação para Cima/genética , Corpo VítreoRESUMO
PURPOSE: To investigate the safety, tolerability, and pharmacokinetics of trabodenoson, a highly selective adenosine mimetic targeting the adenosine A1 receptor. METHODS: In Part 1, 60 healthy adult volunteers were randomized to 14 days of twice-daily topical monocular application of placebo or trabodenoson (200, 400, 800, 1,600, 2,400, or 3,200 µg). In Part 2, 10 subjects were randomized to placebo or 8 escalating doses of bilateral trabodenoson (total daily doses: 1,800-6,400 µg). RESULTS: The incidence of treatment-related adverse events in Part 1 was similar in the trabodenoson (27.8%) and placebo (25.0%) groups. Most were mild in intensity. The most common adverse events (AEs) for trabodenoson and placebo were headache (25.0% vs. 33%, respectively) and eye pain (11.1% vs. 4.2%, respectively). Ocular AEs were infrequent (16.7% and 17.9%, respectively), were self-limited, lasted <24 h, and were typically mild in intensity. The most common ocular AE was eye pain (9.5% and 3.6%, respectively), with a single observation of ocular hyperemia (200 µg trabodenoson). Trabodenoson was rapidly absorbed [median time to maximum concentration (tmax): â¼0.08 to 0.27 h] and eliminated (t½: 0.48-2.0 h), with no evidence of drug accumulation. Systemic exposure to topical trabodenoson was dose related but not dose proportional, with a plateau effect at doses ≥2,400 mg per eye. No clinically significant treatment-related systemic AEs were observed, and increasing systemic exposure had no effect on heart rate or blood pressure. CONCLUSIONS: Ocular doses of trabodenoson up to 3,200 µg per eye were safe and well tolerated in the eye and resulted in no detectable systemic effects in healthy adult volunteers.
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Voluntários Saudáveis , Adulto , Idoso , Relação Dose-Resposta a Droga , Método Duplo-Cego , Olho/fisiopatologia , Humanos , Pessoa de Meia-Idade , Hipertensão Ocular/induzido quimicamenteRESUMO
PURPOSE: The cellular mechanisms linking elevated IOP with glaucomatous damage remain unresolved. Mechanical strains and short-term increases in IOP can trigger ATP release from retinal neurons and astrocytes, but the response to chronic IOP elevation is unknown. As excess extracellular ATP can increase inflammation and damage neurons, we asked if sustained IOP elevation was associated with a sustained increase in extracellular ATP in the posterior eye. METHODS: No ideal animal model of chronic glaucoma exists, so three different models were used. Tg-Myoc(Y437H) mice were examined at 40 weeks, while IOP was elevated in rats following injection of hypertonic saline into episcleral veins and in cynomolgus monkeys by laser photocoagulation of the trabecular meshwork. The ATP levels were measured using the luciferin-luciferase assay while levels of NTPDase1 were assessed using qPCR, immunoblots, and immunohistochemistry. RESULTS: The ATP levels were elevated in the vitreal humor of rats, mice, and primates after a sustained period of IOP elevation. The ecto-ATPase NTPDase1 was elevated in optic nerve head astrocytes exposed to extracellular ATP for an extended period. NTPDase1 was also elevated in the retinal tissue of rats, mice, and primates, and in the optic nerve of rats, with chronic elevation in IOP. CONCLUSIONS: A sustained elevation in extracellular ATP, and upregulation of NTPDase1, occurs in the posterior eye of rat, mouse, and primate models of chronic glaucoma. This suggests the elevation in extracellular ATP may be sustained in chronic glaucoma, and implies a role for altered purinergic signaling in the disease.
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Trifosfato de Adenosina/metabolismo , Antígenos CD/genética , Apirase/genética , Modelos Animais de Doenças , Glaucoma/metabolismo , Pressão Intraocular/fisiologia , Segmento Posterior do Olho/metabolismo , Animais , Antígenos CD/metabolismo , Apirase/metabolismo , Contagem de Células , Doença Crônica , Feminino , Immunoblotting , Imuno-Histoquímica , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ratos , Ratos Endogâmicos BN , Reação em Cadeia da Polimerase em Tempo Real , Células Ganglionares da Retina/patologia , Transdução de SinaisRESUMO
Clinical observations indicate no cataractogenic potential for quetiapine, in contrast to studies in laboratory animals. This randomized, non-inferiority study compared changes in lens opacity during long-term treatment with quetiapine versus risperidone. Patients with schizophrenia or schizoaffective disorder participated in the 2-year, randomized, multicentre, open-label, ophthalmologist-masked, flexible-dose, parallel-group study. Two ophthalmologists examined each patient 6-monthly for presence of nuclear opalescence (N) and cortical (C) or posterior subcapsular opacification (P), according to the lens opacities classification system II. 1098 patients were randomized to treatment. Mean doses were 386.3 mg/day quetiapine and 3.2 mg/day risperidone. Estimated absolute risk differences in cataractogenic events for quetiapine versus risperidone over 2 years were -0.035 (C), -0.012 (N) and -0.017 (P), with upper margins of confidence intervals within the non-inferiority margin of 10%. In post hoc analysis, risk of any lens opacification event was significantly lower for quetiapine than risperidone (6 and 16 events, respectively; risk difference: -0.058; P = 0.035). Efficacy and other safety assessments were in agreement with known profiles of these medications. Quetiapine was non-inferior to risperidone for changes in lens opacity grade in patients with schizophrenia or schizoaffective disorder, indicating that quetiapine does not have clinically significant cataractogenic potential during long-term treatment.
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Antipsicóticos/efeitos adversos , Catarata/induzido quimicamente , Dibenzotiazepinas/efeitos adversos , Transtornos Psicóticos/tratamento farmacológico , Risperidona/efeitos adversos , Esquizofrenia/tratamento farmacológico , Adolescente , Adulto , Idoso , Antipsicóticos/uso terapêutico , Dibenzotiazepinas/uso terapêutico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fumarato de Quetiapina , Risperidona/uso terapêutico , Resultado do Tratamento , Adulto JovemRESUMO
PURPOSE: To compare the power of the 4 intraocular pressure (IOP) measures, that is, peak, mean, range, and SD, over a 24-hour period in predicting IOP variations in order to determine which measure of IOP fluctuation correlates best with actual office-hour readings in glaucoma patients and healthy subjects. METHODS: For this prospective study, 25 subjects with untreated primary open-angle glaucoma and 33 healthy individuals were hospitalized for 24 hours. Measurements of the subjects' IOP for both eyes were recorded with a Goldmann applanation tonometer every 3 hours in the sitting position during the daytime (9 AM to 9 PM) and with a TonoPen in both the sitting and supine positions for 24 hours. Only 1 eye was selected randomly per subject for the final analysis. The strength of association between the estimated values and the actual 24-hour IOP data in habitual body positions was analyzed using the coefficient of determination (R). The differences were calculated. The percentage of subjects with estimated IOP values falling within the cutoff values from the 24-hour data were assessed. RESULTS: The peak IOP was captured outside office hours in 57% of the young subjects, 75% in the elderly control group, and 52% of the glaucoma patients. The estimation of the strength of association for the mean IOP and peak IOP showed strong to moderate correlations (R range from 0.29 to 0.95) compared with the estimation of range and SD of IOP fluctuation, which demonstrated weak to moderate relationships (R range from 0.001 to 0.69). The percentage of significant cases mostly corresponded with the correlation. CONCLUSIONS: With the combination of sitting and supine position readings during office hours, the study provides promising results in estimating the mean and peak IOP in glaucoma patients and healthy subjects; however, it showed little advantage in range and SD of IOP fluctuation.
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Glaucoma de Ângulo Aberto/fisiopatologia , Pressão Intraocular/fisiologia , Postura/fisiologia , Tonometria Ocular/instrumentação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Ritmo Circadiano/fisiologia , Feminino , Glaucoma de Ângulo Aberto/diagnóstico , Gonioscopia , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Visita a Consultório Médico , Estudos Prospectivos , Adulto JovemRESUMO
INTRODUCTION: Nonarteritic anterior ischemic optic neuropathy (NAION), a rare visual disorder, has been reported in men using phosphodiesterase type 5 inhibitors (PDE5i) for erectile dysfunction. AIM: We examined whether intermittent use of PDE5i is associated with acute NAION onset within approximately five half-lives following drug ingestion. METHODS: One hundred two ophthalmology centers in the United States and Europe identified potential cases of NAION. An expert adjudication committee conducted a blind review of the records of those with recent PDE5i use to classify cases as Definite, Possible, or not NAION. Subjects provided information on PDEi use via telephone interview. Each NAION case's PDE5i exposure immediately prior to onset was compared against his recent patterns of use in an observational case-crossover design. A sample size of 40 cases with intermittent PDE5i exposure in the 30 days prior to NAION onset was needed to detect an odds ratio (OR) of 3.0 with 80% power. MAIN OUTCOME MEASURES: The daily relative risk for acute NAION on days within five half-lives of PDE5i use vs. other days was estimated via an OR obtained from conditional logistic regression. RESULTS: Among 43 Definite NAION cases with PDE5i exposure in the prior 30 days, the OR was 2.15 (95% confidence interval [CI]: 1.06, 4.34). When 21 Possible NAION cases were included (n = 64), the OR was 2.36 (95% CI: 1.33, 4.19). CONCLUSIONS: We found an approximately twofold increased risk of acute NAION within five half-lives of PDE5i use compared with use in a more prior time period. Bias from inaccurate recall of exposure was unlikely to have substantially affected the results. Based on our results, we estimate that weekly use of PDE5i adds three NAION cases per 100,000 men 50 years and older annually.
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Disfunção Erétil/tratamento farmacológico , Neuropatia Óptica Isquêmica/induzido quimicamente , Inibidores da Fosfodiesterase 5/efeitos adversos , Idoso , Estudos de Casos e Controles , Disfunção Erétil/epidemiologia , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Neuropatia Óptica Isquêmica/epidemiologia , Neuropatia Óptica Isquêmica/patologia , Inibidores da Fosfodiesterase 5/uso terapêutico , Fatores de Risco , Estados Unidos/epidemiologiaRESUMO
Lysosomes contribute to a multitude of cellular processes, and the pH of the lysosomal lumen plays a central mechanistic role in many of these functions. In addition to controlling the rate of enzymatic degradation for material delivered through autophagic or phagocytotic pathways, lysosomal pH regulates events such as lysosomal fusion with autophagosomes and the release of lysosomal calcium into the cytoplasm. Disruption of either the steady state lysosomal pH or of the regulated manipulations to lysosomal pH may be pathological. For example, chloroquine elevates the lysosomal pH of retinal pigmented epithelial (RPE) cells and triggers a retinopathy characterized by the accumulation of lipofuscin-like material in both humans and animals. Compensatory responses to restore lysosomal pH are observed; new data illustrate that chronic chloroquine treatment increases mRNA expression of the lysosomal/autophagy master transcription factor TcFEB and of the vesicular proton pump vHATPase in the RPE/choroid of mice. An elevated lysosomal pH with upregulation of TcFEB and vHATPase resembles the pathology in fibroblasts of patients with mutant presenilin 1 (PS1), suggesting a common link between age-related macular degeneration (AMD) and Alzheimer's disease. While the absolute rise in pH is often small in these disorders, elevations of only a few tenths of a pH unit can have a major impact on both lysosomal function and the accumulation of waste over decades. Accurate measurement of lysosomal pH can be complex, and imprecise measurements have clouded the field. Protocols to optimize pH measurement from fresh and cultured cells are discussed, and indirect measurements to confirm changes in lysosomal pH and degradative capacity are addressed. The ability of reacidifying treatments to restore degradative function confirms the central role of lysosomal pH in these disorders and identifies potential approaches to treat diseases of lysosomal accumulation like AMD and Alzheimer's disease. In summary, various approaches to determine lysosomal pH in fresh and cultured cells, as well as the potential to restore pH levels to an optimal range, can help identify and repair pathologies associated with lysosomal defects in RPE cells and perhaps also suggest new approaches to treat lysosomal storage diseases throughout the body.
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Células Epiteliais/fisiologia , Lisossomos/fisiologia , Degeneração Retiniana/fisiopatologia , Epitélio Pigmentado da Retina/citologia , Animais , Autofagia/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Epitélio Pigmentado da Retina/patologiaRESUMO
As adenosine 5'-triphosphate (ATP) released from astrocytes can modulate many neural signaling systems, the triggers and pathways for this ATP release are important. Here, the ability of mechanical strain to trigger ATP release through pannexin channels and the effects of sustained strain on pannexin expression were examined in rat optic nerve head astrocytes. Astrocytes released ATP when subjected to 5% of equibiaxial strain or to hypotonic swelling. Although astrocytes expressed mRNA for pannexins 1-3, connexin 43, and VNUT, pharmacological analysis suggested a predominant role for pannexins in mechanosensitive ATP release, with Rho kinase contribution. Astrocytes from panx1(-/-) mice had reduced baseline and stimulated levels of extracellular ATP, confirming the role for pannexins. Swelling astrocytes triggered a regulatory volume decrease that was inhibited by apyrase or probenecid. The swelling-induced rise in calcium was inhibited by P2X7 receptor antagonists A438079 and AZ10606120, in addition to apyrase and carbenoxolone. Extended stretch of astrocytes in vitro upregulated expression of panx1 and panx2 mRNA. A similar upregulation was observed in vivo in optic nerve head tissue from the Tg-MYOC(Y437H) mouse model of chronic glaucoma; genes for panx1, panx2, and panx3 were increased, whereas immunohistochemistry confirmed increased expression of pannexin 1 protein. In summary, astrocytes released ATP in response to mechanical strain, with pannexin 1 the predominant efflux pathway. Sustained strain upregulated pannexins in vitro and in vivo. Together, these findings provide a mechanism by which extracellular ATP remains elevated under chronic mechanical strain, as found in the optic nerve head of patients with glaucoma.
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Trifosfato de Adenosina/metabolismo , Astrócitos/fisiologia , Conexinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Disco Óptico/fisiologia , Estresse Mecânico , Animais , Astrócitos/efeitos dos fármacos , Células Cultivadas , Conexinas/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Glaucoma/fisiopatologia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas de Transporte de Nucleotídeos/metabolismo , Disco Óptico/efeitos dos fármacos , Pressão Osmótica/fisiologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Ratos Long-EvansRESUMO
Healthful cell maintenance requires the efficient degradative processing and removal of waste material. Retinal pigmented epithelial (RPE) cells have the onerous task of degrading both internal cellular debris generated through autophagy as well as phagocytosed photoreceptor outer segments. We propose that the inadequate processing material with the resulting accumulation of cellular waste contributes to the downstream pathologies characterized as age-related macular degeneration (AMD). The lysosomal enzymes responsible for clearance function optimally over a narrow range of acidic pH values; elevation of lysosomal pH by compounds like chloroquine or A2E can impair degradative enzyme activity and lead to a lipofuscin-like autofluorescence. Restoring acidity to the lysosomes of RPE cells can enhance activity of multiple degradative enzymes and is therefore a logical target in early AMD. We have identified several approaches to reacidify lysosomes of compromised RPE cells; stimulation of beta-adrenergic, A2A adenosine and D5 dopamine receptors each lowers lysosomal pH and improves degradation of outer segments. Activation of the CFTR chloride channel also reacidifies lysosomes and increases degradation. These approaches also restore the lysosomal pH of RPE cells from aged ABCA4(-/-) mice with chronically high levels of A2E, suggesting that functional signaling pathways to reacidify lysosomes are retained in aged cells like those in patients with AMD. Acidic nanoparticles transported to RPE lysosomes also lower pH and improve degradation of outer segments. In summary, the ability of diverse approaches to lower lysosomal pH and enhance outer segment degradation support the proposal that lysosomal acidification can prevent the accumulation of lipofuscin-like material in RPE cells.
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Lisossomos/fisiologia , Fagocitose/fisiologia , Segmento Externo das Células Fotorreceptoras da Retina/fisiologia , Epitélio Pigmentado da Retina/fisiologia , Animais , Humanos , Lipofuscina/metabolismo , Imagem ÓpticaRESUMO
Numerous studies have been completed on glaucoma pathogenesis. However, the potential and controversial interaction between ocular biomechanical properties and the glaucomatous diseases process has received much more attention recently. Previous studies have found that collagen tissues gain mutation change in glaucoma patients. This study was conducted to determine the role of collagen in the biomechanics of glaucoma in humans. Its changes may be the result of mechanical modifications brought on by intraocular pressure (IOP) fluctuations. More importantly, biomechanics and genetic evidence indicate that the mutation of collagen may play a role in the process of glaucoma. Alteration of collagen in the outflow pathway may alter mechanical tissue characteristics and a concomitant increase of aqueous humor outflow resistance and elevation of IOP. The variations of collagen, leading to inter-individual differences in scleral and lamina cribrosa properties, result in different susceptibility of individuals to elevated IOP. Therefore, this study hypothesized that collagen mutations may be an original cause of glaucoma.
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Colágeno/genética , Glaucoma/genética , Glaucoma/fisiopatologia , Humanos , Pressão Intraocular , Esclera/patologia , Esclera/fisiopatologia , Malha Trabecular/patologia , Malha Trabecular/fisiopatologiaRESUMO
Lysosomal enzymes function optimally at low pH; as accumulation of waste material contributes to cell aging and disease, dysregulation of lysosomal pH may represent an early step in several pathologies. Here, we demonstrate that stimulation of the P2X7 receptor (P2X7R) for ATP alkalinizes lysosomes in cultured human retinal pigmented epithelial (RPE) cells and impairs lysosomal function. P2X7R stimulation did not kill RPE cells but alkalinized lysosomes by 0.3 U. Receptor stimulation also elevated cytoplasmic Ca(2+); Ca(2+) influx was necessary but not sufficient for lysosomal alkalinization. P2X7R stimulation decreased access to the active site of cathepsin D. Interestingly, lysosomal alkalinization was accompanied by a rise in lipid oxidation that was prevented by P2X7R antagonism. Likewise, the autofluorescence of phagocytosed photoreceptor outer segments increased by lysosomal alkalinization was restored 73% by a P2X7R antagonist. Together, this suggests that endogenous autostimulation of the P2X7R may oxidize lipids and impede clearance. The P2X7R was expressed on apical and basolateral membranes of mouse RPE; mRNA expression of P2X7R and extracellular ATP marker NTPDase1 was raised in RPE tissue from the ABCA4(-/-) mouse model of Stargardt's retinal degeneration. In summary, P2X7R stimulation raises lysosomal pH and impedes lysosomal function, suggesting a possible role for overstimulation in diseases of accumulation.
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Metabolismo dos Lipídeos , Lisossomos/metabolismo , Fagossomos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Cálcio/metabolismo , Bovinos , Linhagem Celular , Membrana Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/genética , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Transcrição GênicaRESUMO
BACKGROUND: The result of primary open-angle glaucoma is the loss of retinal ganglion cells. Transient receptor potential cation channel 6 is a pressure-related channel that may function in the survival of retinal ganglion cells. The purpose of this study was to evaluate the expression levels of the transient receptor potential cation channel 6 gene in patients with primary open-angle glaucoma. DESIGN: Randomization study at Zhongshan Ophthalmic Center in China. PARTICIPANTS: 80 primary open-angle glaucoma patients and 75 cataract patients recruited from Zhongshan Ophthalmic Center. METHODS: Total RNA was extracted from the leukocytes of the peripheral blood collected. The levels of transient receptor potential cation channel 6-messenger RNA were determined by real-time polymerase chain reaction. Related factors including age, intraocular pressure, optic cup-to-disc ratio and visual field defect were analysed accordingly. MAIN OUTCOME MEASURES: Clinical examination and the messenger RNA level. RESULTS: The expression level of the transient receptor potential cation channel 6 gene in the leukocytes of primary open-angle glaucoma patients was two times higher when compared with control cataract patients. The gene expression level was also correlated with intraocular pressure and cup-to-disc ratio. Treatment with different anti-glaucoma drugs did not affect the gene expression. CONCLUSIONS: Increasing expression levels of the transient receptor potential cation channel 6 gene in the blood accompanies chronic elevation of intraocular pressure in primary open-angle glaucoma and may serve as a genetic biomarker for primary open-angle glaucoma.
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Regulação da Expressão Gênica/fisiologia , Marcadores Genéticos , Glaucoma de Ângulo Aberto/genética , Canais de Cátion TRPC/genética , Idoso , Anti-Hipertensivos/uso terapêutico , Feminino , Glaucoma de Ângulo Aberto/diagnóstico , Glaucoma de Ângulo Aberto/tratamento farmacológico , Humanos , Pressão Intraocular/fisiologia , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Canal de Cátion TRPC6 , Tonometria Ocular , Testes de Campo VisualRESUMO
Mitochondrial dysfunction contributing to the pathogenesis of glaucomatous neurodegeneration has stimulated considerable interest recently. In this study, we explored the role of peroxisome proliferator activated receptor-γ co-activator 1α (PGC-1α) in resveratrol-triggered mitochondrial biogenesis for preventing apoptosis in a retinal ganglion cell line RGC-5. Our results showed that serum deprivation induced cell apoptosis in a time-dependent manner. Applying resveratrol maintained the normal mitochondrial membrane potential, decreased the levels of both total and cleaved caspase-3, and inhibited the release of cytochrome c, which subsequently enhanced cell survival. Moreover, resveratrol stimulated mitochondrial biogenesis by increasing the absolute quantity of mitochondria as well as their DNA copies. Treatment with resveratrol promoted the protein expression of SIRT1, but not PGC-1α; instead, resveratrol facilitated PGC-1α translocation from the cytoplasm to the nucleus and up-regulated NRF1 and TFAM, which were blocked by nicotinamide. Collectively, we demonstrate that the SIRT1-dependent PGC-1α subcellular translocation following resveratrol application potentially attenuates serum deprivation-elicited RGC-5 cell death, thereby raising the possibility of mitigating glaucomatous retinopathy by enhancement of mitochondrial biogenesis.
Assuntos
Mitocôndrias/efeitos dos fármacos , Renovação Mitocondrial/efeitos dos fármacos , Células Ganglionares da Retina/efeitos dos fármacos , Estilbenos/farmacologia , Fatores de Transcrição/genética , Animais , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura/química , Citocromos c/metabolismo , Proteínas de Ligação a DNA/agonistas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/agonistas , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Niacinamida/farmacologia , Fator 1 Nuclear Respiratório/agonistas , Fator 1 Nuclear Respiratório/genética , Fator 1 Nuclear Respiratório/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Transporte Proteico/efeitos dos fármacos , Resveratrol , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Transdução de Sinais , Sirtuína 1/genética , Sirtuína 1/metabolismo , Fatores de Transcrição/metabolismoRESUMO
PURPOSE: We evaluated choroidal thickness in the fellow eyes of patients with acute primary angle-closure (APAC) and compared findings to those of normal controls. METHODS: The study group comprised 44 fellow eyes defined as primary angle-closure suspect (PACS) of 44 subjects who had experienced APAC and 43 eyes of 43 healthy volunteers. Using enhanced depth imaging optical coherence tomography (EDI-OCT), the peripapillary and macular choroidal thickness of the PACS eyes and the control eyes were measured and compared at each location or segment. Pearson correlation analysis and a multivariable regression model were used to evaluate the relationships between choroidal thickness and related factors. RESULTS: At all the macular locations, the choroidal thickness was thickest at the subfovea. The PACS eyes had a thicker choroid than the control eyes at all macular locations (all P < 0.05), and it still was significantly thicker after controlling for age, axial length, and sex, except at 3 mm superior from the fovea (P = 0.124). Multivariable linear regression analysis showed that the subfoveal choroidal thickness was significantly thicker in association with the PACS diagnosis, and thinner in association with older subjects and longer axial length eyes. There were no statistically significant differences in the choroidal thickness between the groups at any peripapillary location or segment (P > 0.05). CONCLUSIONS: PACS eyes that had a fellow eye experience of APAC had a thicker macular choroid than the control eyes. The potential role of a thicker choroid as a risk factor for APAC must be investigated further.
Assuntos
Comprimento Axial do Olho/patologia , Corioide/patologia , Glaucoma de Ângulo Fechado/diagnóstico , Aumento da Imagem , Tomografia de Coerência Óptica/métodos , Doença Aguda , Feminino , Glaucoma de Ângulo Fechado/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Acuidade VisualRESUMO
Lysosomal enzymes function optimally in acidic environments, and elevation of lysosomal pH can impede their ability to degrade material delivered to lysosomes through autophagy or phagocytosis. We hypothesize that abnormal lysosomal pH is a key aspect in diseases of accumulation and that restoring lysosomal pH will improve cell function. The propensity of nanoparticles to end up in the lysosome makes them an ideal method of delivering drugs to lysosomes. This study asked whether acidic nanoparticles could traffic to lysosomes, lower lysosomal pH and enhance lysosomal degradation by the cultured human retinal pigmented epithelial cell line ARPE-19. Acidic nanoparticles composed of poly (DL-lactide-co-glycolide) (PLGA) 502 H, PLGA 503 H and poly (DL-lactide) (PLA) colocalized to lysosomes of ARPE-19 cells within 60 min. PLGA 503 H and PLA lowered lysosomal pH in cells compromised by the alkalinizing agent chloroquine when measured 1 hr. after treatment, with acidification still observed 12 days later. PLA enhanced binding of Bodipy-pepstatin-A to the active site of cathepsin D in compromised cells. PLA also reduced the cellular levels of opsin and the lipofuscin-like autofluorescence associated with photoreceptor outer segments. These observations suggest the acidification produced by the nanoparticles was functionally effective. In summary, acid nanoparticles lead to a rapid and sustained lowering of lysosomal pH and improved degradative activity.
Assuntos
Lisossomos/metabolismo , Nanopartículas/química , Nanotecnologia/métodos , Animais , Compostos de Boro/química , Domínio Catalítico , Catepsina D/química , Bovinos , Linhagem Celular , Células Cultivadas , Cloroquina/química , Citometria de Fluxo/métodos , Humanos , Concentração de Íons de Hidrogênio , Immunoblotting , Ácido Láctico/química , Lipofuscina/química , Opsinas/química , Pepstatinas/química , Fagocitose , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros/química , Retina/citologiaRESUMO
Optimal neuronal activity requires that supporting cells provide both efficient nutrient delivery and waste disposal. The incomplete processing of engulfed waste by their lysosomes can lead to accumulation of residual material and compromise their support of neurons. As most degradative lysosomal enzymes function best at an acidic pH, lysosomal alkalinization can impede enzyme activity and increase lipofuscin accumulation. We hypothesize that treatment to reacidify compromised lysosomes can enhance degradation. Here, we demonstrate that degradation of ingested photoreceptor outer segments by retinal pigmented epithelial cells is increased by stimulation of D5 dopamine receptors. D1/D5 receptor agonists reacidified lysosomes in cells alkalinized by chloroquine or tamoxifen, with acidification dependent on protein kinase A. Knockdown with siRNA confirmed acidification was mediated by the D5 receptor. Exposure of cells to outer segments increased lipofuscin-like autofluorescence, but SKF 81297 reduced autofluorescence. Likewise, SKF 81297 increased the activity of lysosomal protease cathepsin D in situ. D5DR stimulation also acidified lysosomes of retinal pigmented epithelial cells from elderly ABCA4(-/-) mice, a model of recessive Stargardt's retinal degeneration. In conclusion, D5 receptor stimulation lowers compromised lysosomal pH, enhancing degradation. The reduced accumulation of lipofuscin-like autofluorescence implies the D5 receptor stimulation may enable cells to better support adjacent neurons.
Assuntos
Agonistas de Dopamina/farmacologia , Células Epiteliais/metabolismo , Lisossomos/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Receptores de Dopamina D5/agonistas , Epitélio Pigmentado da Retina/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Western Blotting , Catepsina D/metabolismo , Bovinos , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Citometria de Fluxo , Fluorescência , Inativação Gênica , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Camundongos , Camundongos Knockout , Pepstatinas , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , RNA Interferente Pequeno , Receptores de Dopamina D1/genética , Receptores de Dopamina D5/genética , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacosRESUMO
The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in lysosomal acidification has been difficult to determine. We demonstrate here that CFTR contributes more to the reacidification of lysosomes from an elevated pH than to baseline pH maintenance. Lysosomal alkalinization is increasingly recognized as a factor in diseases of accumulation, and we previously showed that cAMP reacidified alkalinized lysosomes in retinal pigmented epithelial (RPE) cells. As the influx of anions to electrically balance proton accumulation may enhance lysosomal acidification, the contribution of the cAMP-activated anion channel CFTR to lysosomal reacidification was probed. The antagonist CFTR(inh)-172 had little effect on baseline levels of lysosomal pH in cultured human RPE cells but substantially reduced the reacidification of compromised lysosomes by cAMP. Likewise, CFTR activators had a bigger impact on cells whose lysosomes had been alkalinized. Knockdown of CFTR with small interfering RNA had a larger effect on alkalinized lysosomes than on baseline levels. Inhibition of CFTR in isolated lysosomes altered pH. While CFTR and Lamp1 were colocalized, treatment with cAMP did not increase targeting of CFTR to the lysosome. The inhibition of CFTR slowed lysosomal degradation of photoreceptor outer segments while activation of CFTR enhanced their clearance from compromised lysosomes. Activation of CFTR acidified RPE lysosomes from the ABCA4(-/-) mouse model of recessive Stargardt's disease, whose lysosomes are considerably alkalinized. In summary, CFTR contributes more to reducing lysosomal pH from alkalinized levels than to maintaining baseline pH. Treatment to activate CFTR may thus be of benefit in disorders of accumulation associated with lysosomal alkalinization.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Lisossomos/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Linhagem Celular , Células Cultivadas , AMP Cíclico/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/uso terapêutico , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/genética , Camundongos , Camundongos KnockoutRESUMO
Mechanical deformation produces complex effects on neuronal systems, some of which can lead to dysfunction and neuronal death. While astrocytes are known to respond to mechanical forces, it is not clear whether neurons can also respond directly. We examined mechanosensitive ATP release and the physiological response to this release in isolated retinal ganglion cells. Purified ganglion cells released ATP upon swelling. Release was blocked by carbenoxolone, probenecid or peptide (10)panx, implicating pannexin channels as conduits. Mechanical stretch of retinal ganglion cells also triggered a pannexin-dependent ATP release. Whole cell patch clamp recording demonstrated that mild swelling induced the activation of an Ohmic cation current with linear kinetics. The current was inhibited by removal of extracellular ATP with apyrase, by inhibition of the P2X(7) receptor with A438079, zinc, or AZ 10606120, and by pannexin blockers carbenoxolone and probenecid. Probenecid also inhibited the regulatory volume decrease observed after swelling isolated neurons. Together, these observations indicate mechanical strain triggers ATP release directly from retinal ganglion cells and that this released ATP autostimulates P2X(7) receptors. Since extracellular ATP levels in the retina increase with elevated intraocular pressure, and stimulation of P2X(7) receptors on retinal ganglion cells can be lethal, this autocrine response may impact ganglion cells in glaucoma. It remains to be determined whether the autocrine stimulation of purinergic receptors is a general response to a mechanical deformation in neurons, or whether preventing ATP release through pannexin channels and blocking activation of the P2X(7) receptor, is neuroprotective for stretched neurons.