RESUMEN
[This corrects the article DOI: 10.1155/2017/1763292.].
RESUMEN
INTRODUCTION: Anti-vascular endothelial growth factor (VEGF) agents are used worldwide for advanced-stage diabetic retinopathy (DR). In contrast, apart from blood glucose control, there are no specific treatments that can limit the progression of early-stage DR that starts with pericyte loss and the destruction of the blood-retinal barrier. Here, we examined the efficacy of aflibercept, a potent anti-VEGF agent, against early-DR pathologies in a murine model of streptozotocin (STZ)-induced DR. RESEARCH DESIGN AND METHODS: STZ was intraperitoneally administered in 8-week-old C57BL/6N male mice. After 4 weeks, the mice were divided into aflibercept-treated and saline-treated groups. Eight weeks after the STZ injection, vascular permeability/leakage was measured with fluorescein angiography in live mice. At 4, 6, and 8 weeks after the STZ injection, the eyes were enucleated, flat-mounted, and stained for platelet-derived growth factor receptor-ß to assess pericyte abundance, CD45 to assess leukocyte recruitment, and fluorescein isothiocyanate dextran to assess perfusion. VEGF levels were quantified in each group. The effects of aflibercept on pericyte number, perfusion status, and leukocyte recruitment/accumulation on mice with diabetes retina were evaluated. RESULTS: Our murine model successfully replicated the salient pathologies of DR such as pericytes loss, hyperpermeability, and perfusion blockage. Interestingly, numerous leukocytes and leukocyte clumps were found in diabetic retinal capillaries, especially in the non-perfused border area of the retina, suggesting a possible mechanism for non-perfusion and related pericyte damage. Treatment with aflibercept in mice with diabetes inhibited the upregulation of VEGF and the associated adhesion molecules while reducing the defects in perfusion. Aflibercept also attenuated pericyte loss in the diabetic retina. CONCLUSION: VEGF inhibition through aflibercept treatment decreased leukocyte recruitment and aggregation, perfusion blockage, retinal hypoperfusion, and hyperpermeability in mice with diabetes and ultimately attenuated pericyte loss. Our findings suggest that anti-VEGF strategies may prove useful as possible therapies for limiting the progression of early-stage DR.
Asunto(s)
Diabetes Mellitus Experimental , Pericitos , Animales , Diabetes Mellitus Experimental/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Perfusión , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes de Fusión , Retina , Estreptozocina , Factor A de Crecimiento Endotelial VascularRESUMEN
Purpose: We investigated whether zinc dyshomeostasis, a known mechanism of cell death in acute brain injury, contributes to the activation of matrix metalloproteinases (MMPs) and photoreceptor cell death in experimental retinal detachment (RD). Methods: RD was induced in mice by subretinal injection of 1:1 mixture of balanced salt solution and 1% sodium hyaluronate. On days 1 and 3 post RD, eyeballs were sectioned and examined for cell death (TUNEL staining), the degree of hypoxic insult (Hypoxyprobe staining), free zinc levels (TFL-Zn staining), and MMP-2 and -9 activity (gelatin zymography). In addition, we examined whether modulating extracellular zinc concentration or MMP activation in subretinal fluid affected photoreceptor cell death in RD. These changes were further examined in primary retinal cell and photoreceptor-derived cell (661W) cultures. Results: Photoreceptor cell death peaked on day 3 post RD. Intracellular zinc markedly decreased on day 1 post RD, and subsequently accumulated on day 3. MMP-2 and -9 activity showed a concurrent increase in detached retinas. Detached retinas stained with Hypoxyprobe showed strongly positive cells, especially in the photoreceptor layer. Subretinal injection of a zinc-chelator (CaEDTA) or MMP inhibitor (GM6001, minocycline) at the time of RD significantly attenuated photoreceptor cell death in RD. Similar findings were confirmed in oxygen-glucose-deprived or zinc-exposed cell cultures. Conclusions: Upon RD, hypoxic retinal cells in deep layers underwent zinc dyshomeostasis, MMP activation, and ultimately death. These findings provide new insight into the possible mechanism of photoreceptor death in RD, and as such may prove useful in crafting protective measures for photoreceptor cells.
Asunto(s)
Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Células Fotorreceptoras de Vertebrados/patología , Desprendimiento de Retina/complicaciones , Zinc/metabolismo , Animales , Apoptosis , Células Cultivadas , Modelos Animales de Enfermedad , Activación Enzimática , Homeostasis , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Células Fotorreceptoras de Vertebrados/metabolismo , Desprendimiento de Retina/metabolismo , Desprendimiento de Retina/patología , Líquido Subretiniano/metabolismoRESUMEN
Loss of pericytes, an early hallmark of diabetic retinopathy (DR), results in breakdown of the blood-retinal barrier. Endoplasmic reticulum (ER) stress may be involved in this process. The purpose of this study was to examine the effects of ursodeoxycholic acid (UDCA), a known ameliorator of ER stress, on pericyte loss in DR of streptozotocin- (STZ-) induced diabetic mice. To assess the extent of DR, the integrity of retinal vessels and density of retinal capillaries in STZ-induced diabetic mice were evaluated. Additionally, induction of ER stress and the unfolded protein response (UPR) were assessed in diabetic mice and human retinal pericytes exposed to advanced glycation end products (AGE) or modified low-density lipoprotein (mLDL). Fluorescein dye leakage during angiography and retinal capillary density were improved in UDCA-treated diabetic mice, compared to the nontreated diabetic group. Among the UPR markers, those involved in the protein kinase-like ER kinase (PERK) pathway were increased, while UDCA attenuated UPR in STZ-induced diabetic mice as well as AGE- or mLDL-exposed retinal pericytes in culture. Consequently, vascular integrity was improved and pericyte loss reduced in the retina of STZ-induced diabetic mice. Our findings suggest that UDCA might be effective in protecting against DR.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Retinopatía Diabética/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Pericitos/efectos de los fármacos , Retina/efectos de los fármacos , Ácido Ursodesoxicólico/farmacología , Animales , Barrera Hematorretinal/efectos de los fármacos , Barrera Hematorretinal/metabolismo , Barrera Hematorretinal/patología , Línea Celular , Diabetes Mellitus Experimental/patología , Retinopatía Diabética/patología , Humanos , Ratones , Pericitos/metabolismo , Pericitos/patología , Retina/metabolismo , Retina/patología , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
Loss of pericytes, considered an early hallmark of diabetic retinopathy, is thought to involve abnormal activation of protein kinase C (PKC). We previously showed that the anti-amyotrophic lateral sclerosis (ALS) drug riluzole functions as a PKC inhibitor. Here, we examined the effects of riluzole on pathological changes in diabetic retinopathy. Pathological endpoints examined in vivo included the number of pericytes and integrity of retinal vessels in streptozotocin (STZ)-induced diabetic mice. In addition, PKC activation and the induction of monocyte chemotactic protein (MCP1) were assessed in diabetic mice and in human retinal pericytes exposed to advanced glycation end product (AGE) or modified low-density lipoprotein (mLDL). The diameter of retinal vessels and the number of pericytes were severely reduced, and the levels of MCP1 and PKC were increased in STZ-induced diabetic mice. Administration of riluzole reversed all of these changes. Furthermore, the increased expression of MCP1 in AGE- or mLDL-treated cultured retinal pericytes was inhibited by treatment with riluzole or the PKC inhibitor GF109203X. In silico modeling showed that riluzole fits well within the catalytic pocket of PKC. Taken together, our results demonstrate that riluzole attenuates both MCP1 induction and pericyte loss in diabetic retinopathy, likely through its direct inhibitory effect on PKC.
Asunto(s)
Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Retinopatía Diabética/tratamiento farmacológico , Pericitos/efectos de los fármacos , Riluzol/farmacología , Animales , Retinopatía Diabética/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa C beta/metabolismo , Riluzol/metabolismo , Riluzol/uso terapéutico , EstreptozocinaRESUMEN
BACKGROUND: To evaluate the inhibitory effects of aflibercept on the growth and subretinal invasion of retinoblastoma. METHODS: Xenotransplantation and orthotopic mouse models were created by injecting Y-79 cells subcutaneously and intravitreally, respectively. After induction of retinoblastoma, animals were intraperitoneally injected with aflibercept (25 mg/kg body weight) or saline twice a week for 3 weeks. Tumor size was measured weekly and compared between the two groups. At 4 weeks, animals were sacrificed and an immunohistochemical examination was conducted to compare the microvascular density and degree of apoptosis between groups. In addition, the degree of choroidal invasion was also analyzed in the orthotopic xenotransplantation model. A co-culture system of Y-79 or WERI-Rb-1 cells and human umbilical vein endothelial cells (HUVECs) was used for in vitro experiments, and the anti-angiogenic effect of aflibercept was evaluated by analyzing cell numbers. RESULTS: In the Y-79 xenotransplantation model, aflibercept treatment significantly inhibited tumor growth at 4 weeks versus baseline compared with saline-injected mice (188.53 ± 118.53 mm3 vs. 747.87 ± 118.83 mm3, respectively, P < 0.001). Tumors isolated from aflibercept-treated mice contained fewer blood vessels (8.59 % ± 7.60 % vs. 14.91 % ± 4.53 %, respectively, P < 0.05) and an increased number of apoptotic cells (15.10 ± 9.13 vs. 4.44 ± 2.24, respectively, P < 0.05). In the orthotopic model, the degree of subretinal invasion of tumor cells was significantly reduced after aflibercept treatment (0.07 ± 0.06 vs. 0.15 ± 0.10, P < 0.05). And addition of aflibercept to co-cultures of HUVECs and Y-79, WERI-Rb-1 cells significantly reduced HUVEC proliferation. CONCLUSIONS: Aflibercept reduced retinoblastoma angiogenesis in association with a significant reduction in tumor growth and invasion. These findings suggest that aflibercept could be used in an adjuvant role together with systemic chemotherapy to reduce tumor size and angiogenesis in retinoblastoma.
Asunto(s)
Inhibidores de la Angiogénesis/administración & dosificación , Receptores de Factores de Crecimiento Endotelial Vascular/administración & dosificación , Proteínas Recombinantes de Fusión/administración & dosificación , Neoplasias de la Retina/tratamiento farmacológico , Retinoblastoma/tratamiento farmacológico , Inhibidores de la Angiogénesis/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Técnicas de Cocultivo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inyecciones Intraperitoneales , Ratones , Invasividad Neoplásica , Proteínas Recombinantes de Fusión/farmacología , Neoplasias de la Retina/irrigación sanguínea , Retinoblastoma/irrigación sanguínea , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: To evaluate the effect of metformin on vascular changes in oxygen-induced retinopathy (OIR) in mouse, and to elucidate the possible underlying mechanism. METHODS: OIR mice were treated with metformin by intraperitoneal injection from postnatal day 12 (P12) to P17 or P21. At P17 and P21, vessel formation and avascular areas were assessed using retinal flat mounts. Levels of vascular endothelial growth factor (VEGF) were measured by enzyme-linked immunosorbent assays, and the effects of metformin on VEGF-induced proliferation of human umbilical vein endothelial cells (HUVECs) were assessed. The effects of metformin on the levels of Flk1 (VEGF receptor-2) and phosphorylated Flk1 (pFlk1) were measured by Western blotting (HUVECs) and immunohistochemistry (retinal tissue). RESULTS: Retinal morphologic changes were analyzed between two groups (saline-treated OIR; metformin-treated OIR). Metformin treatment did not change the extent of avascular areas at P17. However, at P21, when OIR pathology was markedly improved in the saline-treated group, OIR pathology still remained in the metformin-treated OIR group. VEGF expression levels did not differ between metformin- and saline-treated OIR groups at P17 and P21, but Flk1 levels were significantly reduced in the metformin group compared with saline-treated OIR group. Moreover, metformin inhibited VEGF-induced cell proliferation and decreased levels of Flk1 and pFlk1, consistent with the interpretation that metformin inhibits vascular growth by reducing Flk1 levels. CONCLUSION: Metformin exerts anti-angiogenesis effects and delays the normal vessel formation in the recovery phase of OIR in mice, likely by suppressing the levels of Flk1.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Metformina/farmacología , Neovascularización Retiniana/tratamiento farmacológico , Retinopatía de la Prematuridad/complicaciones , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Western Blotting , Proliferación Celular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Angiografía con Fluoresceína , Células Endoteliales de la Vena Umbilical Humana , Inmunohistoquímica , Inyecciones Intraperitoneales , Metformina/administración & dosificación , Ratones , Retina/efectos de los fármacos , Retina/patología , Neovascularización Retiniana/etiología , Retinopatía de la Prematuridad/tratamiento farmacológicoRESUMEN
Metallothionein-3 (Mt3), a zinc (Zn)-regulatory protein mainly expressed in the central nervous system, may contribute to oxidative cell death. In the present study, we examined the possible role of Mt3 in streptozotocin (STZ)-induced islet cell death and consequent hyperglycemia. Quantitative real-time polymerase chain reaction (RT-PCR) confirmed that islet cells expressed Mt3 mRNA. In all cases, wild-type (WT) mice injected with STZ exhibited hyperglycemia 7-21 days later. In stark contrast, all Mt3-null mice remained normoglycemic following STZ injection. STZ treatment increased free Zn levels in islet cells and induced their death in WT mice, but failed to do so in Mt3-null mice. Consistent with this, cultured Mt3-null islet cells exhibited striking resistance to STZ toxicity. Notably, PDE3a (phosphodiesterase 3A) was downregulated in islets of Mt3-null mice compared to those of WT mice, and was not induced by STZ treatment. Moreover, the PDE3 inhibitor cilostazol reduced islet cell death, likely by increasing cAMP levels, further supporting a role for PDE3 in STZ-induced islet cell death. Collectively, these results demonstrate that Mt3 may act through PDE3a to play a key role in Zn dyshomeostasis and cell death in STZ-treated islets.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Proteínas del Tejido Nervioso/metabolismo , Animales , Muerte Celular , Línea Celular , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Metalotioneína 3 , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Nitroprusiato/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estreptozocina , Zinc/metabolismoRESUMEN
Eukaryotic algae and cyanobacteria produce hydrogen under anaerobic and limited aerobic conditions. Here we show that novel microalgal strains (Chlorella vulgaris YSL01 and YSL16) upregulate the expression of the hydrogenase gene (HYDA) and simultaneously produce hydrogen through photosynthesis, using CO2 as the sole source of carbon under aerobic conditions with continuous illumination. We employ dissolved oxygen regimes that represent natural aquatic conditions for microalgae. The experimental expression of HYDA and the specific activity of hydrogenase demonstrate that C. vulgaris YSL01 and YSL16 enzymatically produce hydrogen, even under atmospheric conditions, which was previously considered infeasible. Photoautotrophic H2 production has important implications for assessing ecological and algae-based photolysis.
Asunto(s)
Chlorella vulgaris/metabolismo , Hidrógeno/metabolismo , Microalgas/metabolismo , Fotosíntesis , Aerobiosis , Procesos Autotróficos , Oxígeno/metabolismoRESUMEN
In the present study, we investigated possible roles of the zinc (Zn)-binding protein metallothionein-3 (MT3) and cellular Zn in a mouse model of laser-induced choroidal neovascularization (CNV) using wild-type (WT) and MT3-knockout (KO) mice. Quantitative RT-PCR was used for the detection of MT3 mRNA. CNV was induced in mice between 8 and 12 weeks of age by disrupting the Bruch's membrane using an argon laser. Fundus photography and fluorescein angiography (FA) were performed 2 weeks following laser photocoagulation. The possible connection between MT3 and vascular endothelial growth factor (VEGF) expression was explored by quantifying VEGF levels in WT and MT3-KO mouse retinas by enzyme-linked immunosorbent assay. The role of Zn in VEGF expression was tested in WT and MT3-KO cells treated with pyrithione, with or without additional Zn, using immunoblotting and fluorescence photomicrography. Following laser-treatment, MT3-KO mice exhibited substantially smaller areas of CNV compared to WT mice. In addition, retinal angiograms revealed less severe fluorescein leakage in MT3-KO mice than in WT mice. On day 14 following the induction of CNV, VEGF expression was markedly increased in WT mice, but remained unchanged in MT3-KO mice. Consistent with the possible involvement of Zn released from MT3, raising intracellular Zn levels increased VEGF levels and activated its receptor, Flk-1, in both WT and MT3-KO retinal cells. Present results demonstrated that neural retinal cells express high levels of MT3, which might play a role in the process of CNV development. Moreover, Zn released from MT3 may contribute to VEGF induction.
Asunto(s)
Neovascularización Coroidal/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Astrocitos/metabolismo , Astrocitos/patología , Neovascularización Coroidal/diagnóstico por imagen , Neovascularización Coroidal/patología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Angiografía con Fluoresceína , Regulación de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Immunoblotting , Rayos Láser , Masculino , Metalotioneína 3 , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Radiografía , Retina/metabolismo , Retina/patología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Zinc/metabolismoRESUMEN
BACKGROUND: Microalgal biomass contains a high level of carbohydrates which can be biochemically converted to biofuels using state-of-the-art strategies that are almost always needed to employ a robust pretreatment on the biomass for enhanced energy production. In this study, we used an ultrasonic pretreatment to convert microalgal biomass (Scenedesmus obliquus YSW15) into feasible feedstock for microbial fermentation to produce ethanol and hydrogen. The effect of sonication condition was quantitatively evaluated with emphases on the characterization of carbohydrate components in microalgal suspension and on subsequent production of fermentative bioenergy. METHOD: Scenedesmus obliquus YSW15 was isolated from the effluent of a municipal wastewater treatment plant. The sonication durations of 0, 10, 15, and 60 min were examined under different temperatures at a fixed frequency and acoustic power resulted in morphologically different states of microalgal biomass lysis. Fermentation was performed to evaluate the bioenergy production from the non-sonicated and sonicated algal biomasses after pretreatment stage under both mesophilic (35°C) and thermophilic (55°C) conditions. RESULTS: A 15 min sonication treatment significantly increased the concentration of dissolved carbohydrates (0.12 g g(-1)), which resulted in an increase of hydrogen/ethanol production through microbial fermentation. The bioconvertibility of microalgal biomass sonicated for 15 min or longer was comparable to starch as a control, indicating a high feasibility of using microalgae for fermentative bioenergy production. Increasing the sonication duration resulted in increases in both algal surface hydrophilicity and electrostatic repulsion among algal debris dispersed in aqueous solution. Scanning electron microscope images supported that ruptured algal cell allowed fermentative bacteria to access the inner space of the cell, evidencing an enhanced bioaccessibility. Sonication for 15 min was the best for fermentative bioenergy (hydrogen/ethanol) production from microalga, and the productivity was relatively higher for thermophilic (55°C) than mesophilic (35°C) condition. CONCLUSION: These results demonstrate that more bioavailable carbohydrate components are produced through the ultrasonic degradation of microalgal biomass, and thus the process can provide a high quality source for fermentative bioenergy production.
RESUMEN
PURPOSE: We investigated the mechanism of tamoxifen (TAM) retinotoxicity using human retinal pigment epithelial (RPE)-derived (ARPE-19) and photoreceptor-derived (661W) cells. METHODS: Cultured ARPE-19 and 661W cells were treated with 5 to 10 µM TAM, and the resultant cell death was quantified using lactate dehydrogenase (LDH) release assay. Cellular oxidative stress was determined by measuring 5-(and-6)-carboxy-2',7'-dichlorohydrofluorescein diacetate (H(2)-DCFDA) fluorescence. Changes in intracellular free zinc levels were monitored using the zinc-specific fluorescent dye, FluoZin-3 AM. Autophagic vacuole formation was assessed morphologically in ARPE-19 and 661W cells transfected with the fluorescent protein-conjugated markers, RFP-LC3 or GFP-LC3. RESULTS: Following exposure to TAM, both ARPE-19 and 661W cells had cytosolic vacuoles within 1 hour and underwent cell death within 18 hours. In both cell types, TAM-induced cell death was accompanied by increased oxidative stress and elevated zinc levels, and was attenuated by the antioxidant N-acetyl-L-cysteine (NAC) or the zinc chelator N,N,N'N'-tetrakis(-)(2-pyridylmethyl)-ethylenediamine (TPEN). The levels of LC3-II as well as the number of autophagic vacuoles (AVs) increased after TAM treatment. Double staining for lysosomes and AVs showed that autolysosome formation proceeded normally. Consistent with this, autophagy flux was increased. Finally, as shown in other cases of autophagic cell death, lysosomal membrane permeabilization (LMP) as well as caspase-dependent apoptosis contributed to TAM-induced cell death. CONCLUSIONS: ARPE-19 and 661W cells were vulnerable similarly to TAM-induced cytotoxicity. Increases in zinc levels and oxidative stress, excessive activation of autophagy flux, and ultimately the occurrence of LMP and consequent caspase activation may contribute to the well-established retinal cytotoxicity of TAM.
Asunto(s)
Autofagia/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/patología , Epitelio Pigmentado de la Retina/patología , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/farmacología , Células Cultivadas , Citosol/patología , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Vacuolas/patología , Zinc/metabolismoRESUMEN
Anaerobic fermentation for hydrogen (H2) production was studied in a two-stage fermentation system fed with different ripened fruit feedstocks (apple, pear, and grape). Among the feedstocks, ripened apple was the most efficient substrate for cumulative H2 production (4463.7 mL-H2 L(-1)-culture) with a maximum H2 yield (2.2 mol H2 mol(-1) glucose) in the first stage at a hydraulic retention time (HRT) of 18 h. The additional cumulative biohydrogen (3337.4 mL-H2 L(-1)-culture) was produced in the second stage with the reused residual substrate from the first stage. The major byproducts in this study were butyrate, acetate, and ethanol, and butyrate was dominant among them in all test runs. During the two-stage system, the energy efficiency (H(2) conversion) obtained from mixed ripened fruits (RF) increased from 4.6% (in the first stage) to 15.5% (in the second stage), which indicated the energy efficiency can be improved by combined hydrogen production process. The RF could be used as substrates for biohydrogen fermentation in a two-stage (dark/dark) fermentation system.
Asunto(s)
Biotecnología/métodos , Oscuridad , Fuentes Generadoras de Energía , Fermentación , Frutas/química , Frutas/crecimiento & desarrollo , Hidrógeno/análisis , Metabolismo de los Hidratos de Carbono , Etanol/análisis , Ácidos Grasos Volátiles/análisis , Estudios de Factibilidad , Aguas del Alcantarillado/química , Factores de Tiempo , Residuos/análisisRESUMEN
PURPOSE: To characterize and investigate the mechanism of chloroquine (CQ) retinotoxicity in human retinal pigment epithelium-derived ARPE-19 cells. METHODS: Cultured ARPE-19 cells were exposed to 10 to 250 µM CQ, and cell death was quantified using a lactate dehydrogenase release assay. Autophagy was studied in ARPE-19 cells transfected with GFP-LC3. Lysosomes in living cells were stained and observed by live-cell confocal microscopy. RESULTS: After exposure to CQ, ARPE-19 cells developed cytosolic vacuoles within 1 hour and underwent cell lysis within 24 hours. The levels of LC3-II, beclin-1 and, p62, as well as the number GFP-LC3- and RPF-LC3-positive autophagic vacuoles (AVs), increased after CQ treatment, indicating that autophagy was activated. However, lysosomal staining revealed that almost all AVs were separate from lysosomes; thus, fusion between AVs and lysosomes was completely blocked. In addition, the levels of ubiquitinated proteins and GFP-mHttp aggregates in ARPE-19 cells were increased by CQ, providing further evidence that autophagic degradation was inhibited. CONCLUSIONS: CQ induces vacuole formation and cell death in ARPE-19 cells. Initially, vacuoles developed from enlarged lysosomes, followed by the activation of upstream steps in the autophagy pathway and the formation of LC3-positive AVs. Because CQ blocked the fusion of AVs with lysosomes, autophagic protein degradation was inhibited, indicating that CQ-induced retinotoxicity may be caused by the accumulation of potentially toxic ubiquitinated proteins.
Asunto(s)
Antimaláricos/toxicidad , Autofagia/efectos de los fármacos , Cloroquina/toxicidad , Lisosomas/efectos de los fármacos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Beclina-1 , Caspasa 3/metabolismo , Muerte Celular , Células Cultivadas , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas Fluorescentes Verdes/genética , Humanos , Immunoblotting , L-Lactato Deshidrogenasa/metabolismo , Lisosomas/patología , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Especies Reactivas de Oxígeno/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Proteína Sequestosoma-1 , Transfección , Vacuolas/efectos de los fármacos , Vacuolas/patologíaRESUMEN
The effect of chemical oxygen demand/sulfate (COD/SO(4)(2-)) ratio on fermentative hydrogen production using enriched mixed microflora has been studied. The chemostat system maintained with a substrate (glucose) concentration of 15 g COD L(-1) exhibited stable H(2) production at inlet sulfate concentrations of 0-20 g L(-1) during 282 days. The tested COD/SO(4)(2-) ratios ranged from 150 to 0.75 (with control) at pH 5.5 with hydraulic retention time (HRT) of 24, 12 and 6h. The hydrogen production at HRT 6h and pH 5.5 was not influenced by decreasing the COD/SO(4)(2-) ratio from 150 to 15 (with control) followed by noticeable increase at COD/SO(4)(2-) ratios of 5 and 3, but it was slightly decreased when the COD/SO(4)(2-) ratio further decreased to 1.5 and 0.75. These results indicate that high sulfate concentrations (up to 20,000 mg L(-1)) would not interfere with hydrogen production under the investigated experimental conditions. Maximum hydrogen production was 2.95, 4.60 and 9.40 L day(-1) with hydrogen yields of 2.0, 1.8 and 1.6 mol H(2) mol(-1) glucose at HRTs of 24, 12 and 6h, respectively. The volatile fatty acid (VFA) fraction produced during the reaction was in the order of butyrate>acetate>ethanol>propionate in all experiments. Fluorescence In Situ Hybridization (FISH) analysis indicated the presence of Clostridium spp., Clostridium butyricum, Clostridium perfringens and Ruminococcus flavefaciens as hydrogen producing bacteria (HPB) and absence of sulfate reducing bacteria (SRB) in our study.