RESUMO
Age-related macular degeneration (AMD) is associated with formation of drusen, clusters of lipids, and oxidized lipid products under the retinal pigment epithelium (RPE). 7-Ketocholesterol (7KC) is a form of oxidized cholesterol present in drusen and is hypothesized to play a role in AMD pathogenesis. The association of 7KC with cellular toxicity and inflammation, key elements of AMD pathology, has been demonstrated. However, the effects of 7KC on altering RPE bioenergetics, a potentially important pathologic process in AMD, are unclear. Herein, we describe the effects of non-lethal doses of 7KC on the bioenergetics and phenotype of RPE cells in culture. Metabolic analysis demonstrated a significant dose-dependent increase in total ATP production rates that was driven primarily by an increase in glycolysis. The increase in glycolysis was accompanied by an increase in glucose uptake and increased expression of hexokinase 1. Increased levels of Translocase of Outer Mitochondrial Membrane 20 and NADH:Ubiquinone Oxidoreductase Core Subunit S1, Succinate dehydrogenase, Ubiquinol-Cytochrome C Reductase Core Protein 2, Cytochrome C Oxidase II, and ATP synthase subunit beta, proteins involved in oxidative phosphorylation (OXPHOS), were also seen. However, specific electron transport chain activity remained unchanged. 7KC-treated cells also demonstrated a change in cellular morphology with decreased expression of epithelial markers. In summary, 7KC has significant effects on the bioenergetics and morphology of RPE cells reflective of findings seen in clinical AMD.
Assuntos
Degeneração Macular , Epitélio Pigmentado da Retina , Humanos , Epitélio Pigmentado da Retina/metabolismo , Retina/metabolismo , Metabolismo Energético , Degeneração Macular/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
Increased dietary consumption of docosahexaenoic acid (DHA) is associated with decreased risk for Alzheimer's disease (AD). These effects have been postulated to arise from DHA's pleiotropic effects on AD pathophysiology, including its effects on ß-amyloid (Aß) production, aggregation, and toxicity. While in vitro studies suggest that DHA may inhibit and reverse the formation of toxic Aß oligomers, it remains uncertain whether these mechanisms operate in vivo at the physiological concentrations of DHA attainable through dietary supplementation. We sought to clarify the effects of dietary DHA supplementation on Aß indices in a transgenic APP/PS1 rat model of AD. Animals maintained on a DHA-supplemented diet exhibited reductions in hippocampal Aß plaque density and modest improvements on behavioral testing relative to those maintained on a DHA-depleted diet. However, DHA supplementation also increased overall soluble Aß oligomer levels in the hippocampus. Further quantification of specific conformational populations of Aß oligomers indicated that DHA supplementation increased fibrillar (i.e. putatively less toxic) Aß oligomers and decreased prefibrillar (i.e. putatively more toxic) Aß oligomers. These results provide in vivo evidence suggesting that DHA can modulate Aß aggregation by stabilizing soluble fibrillar Aß oligomers and thus reduce the formation of both Aß plaques and prefibrillar Aß oligomers. However, since fibrillar Aß oligomers still retain inherent neurotoxicity, DHA may need to be combined with other interventions that can additionally reduce fibrillar Aß oligomer levels for more effective prevention of AD in clinical settings.
Assuntos
Doença de Alzheimer/dietoterapia , Peptídeos beta-Amiloides/metabolismo , Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos , Hipocampo/metabolismo , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/dietoterapia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/psicologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Hipocampo/patologia , Humanos , Masculino , Aprendizagem em Labirinto , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Placa Amiloide/psicologia , Presenilina-1/genética , Presenilina-1/metabolismo , Multimerização Proteica , Ratos Sprague-Dawley , Ratos Transgênicos , Resultado do TratamentoRESUMO
Age-related macular degeneration (AMD) is one of the major causes of vision loss in the elderly in most developed countries. Among other causes, oxidative stress in the retinal pigment epithelium (RPE) has been hypothesized to be a major driving force of AMD pathology. Oxidative stress could be treated by antioxidant administration into the RPE cells. However, to achieve high in-vivo efficacy of an antioxidant, it is imperative that the agent be able to penetrate the tissues and cells. Evidence suggests that lipophilicity governs cellular penetrance. Out of many antioxidant candidates, N-acetyl-L-cysteine (a prodrug of L-cysteine) (NAC) is a potent antioxidant as the bioavailability of the parent drug, L-cysteine, determines the production of glutathione; the universal antioxidant that regulates ROS. To increase the lipophilicity, four ester derivatives of N-acetylcysteine: N-acetylcysteine methyl ester, N-acetylcysteine ethyl ester, N-acetylcysteine propyl ester, and N-acetylcysteine butyl ester were synthesized. To mimic in vitro AMD conditions, hydroquinone, a component of cigarette smoke, was used as the oxidative insult. Cytosolic and mitochondrial protection against oxidative stress were tested using cytosolic and mitochondrial specific assays. The results provide evidence that these lipophilic cysteine prodrugs provide increased protection against oxidative stress in human RPE cells compared with NAC.
Assuntos
Pró-Fármacos , Idoso , Células Epiteliais , Humanos , Estresse Oxidativo , Pró-Fármacos/farmacologia , Epitélio Pigmentado da Retina , Pigmentos da RetinaRESUMO
Purpose: This study investigated the effects of esterification and increased lipophilicity on cellular penetration, accumulation and retention in ARPE-19-nic cells using ester functionalized rhodamine B dyes. Methods: Rhodamine B was esterified to generate four dyes with increasing lipophilicity. Cellular uptake, retention and mitochondrial localization were investigated in vitro using ARPE-19-nic cells using direct intracellular and extracellular and mitochondrial fluorescence quantitation, confocal and high-resolution live cell imaging and co-localization with Mito-GFP. Results: Cellular penetrance, mitochondrial accumulation, and retention of the esterified dyes were increased in ARPE-19-nic cells compared with the nonesterified parent dye by direct fluorescence quantitation. Imaging demonstrated intracellular accumulation was confined to mitochondria as confirmed by colocalization with Mito-GFP. Conclusions: Esterification is an effective way to increase lipophilicity of a dye to improve cellular penetration of chemical entities. These observations may be key to improving retinal drug delivery for retinal pigment epithelium-based diseases. Translational Relevance: Understanding the intracellular distribution of drugs into retinal pigment epithelium cells is a critical component for identifying potential therapies for retinal pigment epithelium-based diseases.
Assuntos
Epitélio Pigmentado da Retina , Pigmentos da Retina , Transporte Biológico , Esterificação , RodaminasRESUMO
PURPOSE: Docosahexaenoic acid (DHA) was supplemented in a single-site, placebo-controlled, randomized clinical trial designed to slow vision loss associated with X-linked retinitis pigmentosa (XLRP); the DHAX Trial. We previously reported no significant differences between supplemented and placebo groups in intent-to-treat analysis of primary ERG outcomes. Assessed herein are hypothesis-generating measures of ancillary visual function outcomes in participants fully adhering to trial protocol. METHODS: Male participants with XLRP (range, 7-31 years) received 30 mg DHA/kg/d (n = 29) or placebo (n = 22) for 4 years. Visual outcomes were measured annually and red blood cell (RBC) DHA determined every 6 months. RESULTS: Oral DHA supplementation increased mean RBC-DHA levels by 4-fold (P < 0.0001) over placebo. No group differences in progression were found for visual acuity (P = 0.11), shape discrimination (P = 0.18), or fundus appearance (P = 0.70). Optical coherence tomography (OCT) became available during year 2 of the trial; no group differences were seen in ellipsoid zone constriction (P = 0.87) over 2 years. Yearly rates of progression were reduced for dark-adapted thresholds (P = 0.06) and visual field sensitivity for foveal, macular, peripheral, total, and ellipsoid zone regions by DHA supplementation (P = 0.039, P = 0.031, P < 0.0001, P < 0.0001, and P = 0.033). Rates of visual field sensitivity decline were dependent on RBC-DHA (P = 0.046 to <0.0001). CONCLUSIONS: Supplementation of DHA significantly elevated blood DHA levels and reduced the rate of progression in final dark-adapted thresholds and visual field sensitivity. From the relationship between RBC-DHA and the rate of field sensitivity loss, we can extrapolate that an RBC-DHA level of 17% could minimize the decline in field sensitivity. (ClinicalTrials.gov number, NCT00100230.)
Assuntos
Ácidos Docosa-Hexaenoicos/uso terapêutico , Doenças Genéticas Ligadas ao Cromossomo X/tratamento farmacológico , Retinose Pigmentar/tratamento farmacológico , Adolescente , Adulto , Criança , Progressão da Doença , Percepção de Forma/efeitos dos fármacos , Fundo de Olho , Humanos , Masculino , Retinose Pigmentar/genética , Campos Visuais/efeitos dos fármacos , Adulto JovemRESUMO
IMPORTANCE: X-linked retinitis pigmentosa is a severe inherited retinal degenerative disease with a frequency of 1 in 100,000 persons. Because no cure is available for this orphan disease and treatment options are limited, slowing of disease progression would be a meaningful outcome. OBJECTIVE: To determine whether high-dose docosahexaenoic acid (DHA), an ω-3 polyunsaturated fatty acid, slows progression of X-linked retinitis pigmentosa measured by cone electroretinography (ERG). DESIGN, SETTING, AND PARTICIPANTS: A 4-year, single-site, randomized, placebo-controlled, double-masked phase 2 clinical trial at a research center specializing in medical retina. Seventy-eight male patients diagnosed as having X-linked retinitis pigmentosa were randomized to DHA or placebo. Data were omitted for 2 patients with non-X-linked retinitis pigmentosa and 16 patients who were unable to follow protocol during the first year. The remaining participants were tested annually and composed a modified intent-to-treat cohort (DHA group, n = 33; placebo group, n = 27). INTERVENTIONS: All participants received a multivitamin and were randomly assigned to oral DHA (30 mg/kg/d) or placebo. MAIN OUTCOMES AND MEASURES: The primary outcome was the rate of loss of cone ERG function. Secondary outcomes were rod and maximal ERG amplitudes and cone ERG implicit times. Capsule counts and red blood cell DHA levels were assessed to monitor adherence. RESULTS: Average (6-month to 4-year) red blood cell DHA levels were 4-fold higher in the DHA group than in the placebo group (P < .001). There was no difference between the DHA and placebo groups in the rate of cone ERG functional loss (0.028 vs 0.022 log µV/y, respectively; P = .30). No group differences were evident for change in rod ERG (P = .27), maximal ERG (P = .65), or cone implicit time (no change over 4 years). The rate of cone loss (ie, event rate) was markedly reduced compared with rates in previous studies. No severe treatment-emergent adverse events were found. CONCLUSIONS AND RELEVANCE: Long-term DHA supplementation was not effective in slowing the loss of cone or rod ERG function associated with X-linked retinitis pigmentosa. Participant dropout and lower-than-expected disease event rate limited power to detect statistical significance. A larger sample size, longer trial, and attainment of a target blood DHA level (13%) would be desirable. While DHA supplementation at 30 mg/kg/d does not present serious adverse effects, routine monitoring of gastrointestinal tolerance is prudent. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00100230.
Assuntos
Ácidos Docosa-Hexaenoicos/administração & dosagem , Doenças Genéticas Ligadas ao Cromossomo X/tratamento farmacológico , Retinose Pigmentar/tratamento farmacológico , Administração Oral , Adolescente , Adulto , Cápsulas , Criança , Cromatografia Gasosa , Progressão da Doença , Ácidos Docosa-Hexaenoicos/sangue , Ácidos Docosa-Hexaenoicos/uso terapêutico , Método Duplo-Cego , Eletrorretinografia , Membrana Eritrocítica/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Doenças Genéticas Ligadas ao Cromossomo X/fisiopatologia , Humanos , Masculino , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Retinose Pigmentar/diagnóstico , Retinose Pigmentar/fisiopatologia , Resultado do Tratamento , Adulto JovemRESUMO
PURPOSE: Docosahexaenoic acid (DHA) continues to be evaluated and recommended as treatment and prophylaxis for various diseases. We recently assessed efficacy of high-dose DHA supplementation to slow vision loss in patients with X-linked retinitis pigmentosa (XLRP) in a randomized clinical trial. Because DHA is a highly unsaturated fatty acid, it could serve as a target for free-radical induced oxidation, resulting in increased oxidative stress. Biosafety was monitored during the 4-year trial to determine whether DHA supplementation was associated with identifiable risks. METHODS: Males (n = 78; 7-31 years) meeting entry criteria were enrolled. The modified intent-to-treat cohort (DHA = 33; placebo = 27) adhered to the protocol ≥ 1 year. Participants were randomized to an oral dose of 30 mg/kg/d DHA or placebo plus a daily multivitamin. Comprehensive metabolic analyses were assessed for group differences. Treatment-emergent adverse events including blood chemistry metabolites were recorded. RESULTS: By year 4, supplementation elevated plasma and red blood cell-DHA 4.4- and 3.6-fold, respectively, compared with the placebo group (P < 0.00001). Over the trial duration, no significant differences between DHA and placebo groups were found for vitamin A, vitamin E, platelet aggregation, antioxidant activity, lipoprotein cholesterol, or oxidized LDL levels (all P > 0.14). Adverse events were transient and not considered severe (e.g., gastrointestinal [GI] irritability, blood chemistry alterations). One participant was unable to tolerate persistent GI discomfort. CONCLUSIONS: Long-term, high-dose DHA supplementation to patients with XLRP was associated with limited safety risks in this 4-year trial. Nevertheless, GI symptoms should be monitored in all patients taking high dose DHA especially those with personal or family history of GI disturbances. (ClinicalTrials.gov number, NCT00100230.).