RESUMEN
Age-related macular degeneration (AMD) is a severe retinal eye disease where dysfunctional mitochondria and damaged mitochondrial DNA in retinal pigment epithelium (RPE) have been demonstrated to underlie the pathogenesis of this devastating disease. In the present study, we aimed to examine whether damaged mitochondria induce inflammasome activation in human RPE cells. Therefore, ARPE-19 cells were primed with IL-1α and exposed to the mitochondrial electron transport chain complex III inhibitor, antimycin A. We found that antimycin A-induced mitochondrial dysfunction caused caspase-1-dependent inflammasome activation and subsequent production of mature IL-1ß and IL-18 in human RPE cells. AIM2 and NLRP3 appeared to be the responsible inflammasome receptors upon antimycin A-induced mitochondrial damage. We aimed at verifying our findings using hESC-RPE cells but antimycin A was absorbed by melanin. Therefore, results were repeated on D407 RPE cell cultures. Antimycin A-induced mitochondrial and NADPH oxidase-dependent ROS production occurred upstream of inflammasome activation, whereas K+ efflux was not required for inflammasome activation in antimycin A-treated human RPE cells. Collectively, our data emphasize that dysfunctional mitochondria regulate the assembly of inflammasome multiprotein complexes in the human RPE cells. The present study associates AIM2 with the pathogenesis of AMD.
Asunto(s)
Antimicina A/farmacología , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Inflamasomas/genética , Degeneración Macular/genética , Mitocondrias/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Línea Celular , Proteínas de Unión al ADN/biosíntesis , Humanos , Inflamasomas/efectos de los fármacos , Inflamasomas/metabolismo , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/metabolismo , Mitocondrias/metabolismo , ARN/genética , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/patología , Transducción de SeñalRESUMEN
DNA damage accumulates in aged postmitotic retinal pigment epithelium (RPE) cells, a phenomenon associated with the development of age-related macular degeneration. In this study, we have experimentally induced DNA damage by ultraviolet B (UVB) irradiation in interleukin-1α (IL-1α)-primed ARPE-19 cells and examined inflammasome-mediated signaling. To reveal the mechanisms of inflammasome activation, cells were additionally exposed to high levels of extracellular potassium chloride, n-acetyl-cysteine, or mitochondria-targeted antioxidant MitoTEMPO, prior to UVB irradiation. Levels of interleukin-18 (IL-18) and IL-1ß mRNAs were detected with qRT-PCR and secreted amounts of IL-1ß, IL-18, and caspase-1 were measured with ELISA. The role of nucleotide-binding domain and leucine-rich repeat pyrin containing protein 3 (NLRP3) in UVB-induced inflammasome activation was verified by using the NLRP3-specific siRNA. Reactive oxygen species (ROS) levels were measured immediately after UVB exposure using the cell-permeant 2',7'-dichlorodihydrofluorescein diacetate (H2 DCFDA) indicator, the levels of cyclobutane pyrimidine dimers were assayed by cell-based ELISA, and the extracellular levels of adenosine triphosphate (ATP) determined using a commercial bioluminescence assay. We found that pro-IL-18 was constitutively expressed by ARPE-19 cells, whereas the expression of pro-IL-1ß was inducible by IL-1α priming. UVB induced the release of mature IL-18 and IL-1ß but NLRP3 contributed only to the secretion of IL-1ß. At the mechanistic level, the release of IL-1ß was regulated by K+ efflux, whereas the secretion of IL-18 was dependent on ROS production. As well as K+ efflux, the cells released ATP following UVB exposure. Collectively, our data suggest that UVB clearly stimulates the secretion of mature IL-18 as a result of ROS induction, and this response is associated with DNA damage. Moreover, in human RPE cells, K+ efflux mediates the UVB-activated NLRP3 inflammasome signaling, leading to the processing of IL-1ß.
Asunto(s)
Inflamasomas/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Rayos Ultravioleta , Daño del ADN , Reparación del ADN , Humanos , Inflamasomas/inmunología , Inflamasomas/efectos de la radiación , Especies Reactivas de Oxígeno/metabolismo , Epitelio Pigmentado de la Retina/inmunología , Epitelio Pigmentado de la Retina/efectos de la radiación , Transducción de SeñalRESUMEN
Inflammation is a key underlying factor of age-related macular degeneration (AMD) and inflammasome activation has been linked to disease development. Induced pluripotent stem-cell-derived retinal pigment epithelial cells (iPSC-RPE) are an attractive novel model system that can help to further elucidate disease pathways of this complex disease. Here, we analyzed the effect of dysfunctional protein clearance on inflammation and inflammasome activation in iPSC-RPE cells generated from a patient suffering from age-related macular degeneration (AMD) and an age-matched control. We primed iPSC-RPE cells with IL-1α and then inhibited both proteasomal degradation and autophagic clearance using MG-132 and bafilomycin A1, respectively, causing inflammasome activation. Subsequently, we determined cell viability, analyzed the expression levels of inflammasome-related genes using a PCR array, and measured the levels of pro-inflammatory cytokines IL-1ß, IL-6, IL-8, and MCP-1 secreted into the medium. Cell treatments modified the expression of 48 inflammasome-related genes and increased the secretion of mature IL-1ß, while reducing the levels of IL-6 and MCP-1. Interestingly, iPSC-RPE from an AMD donor secreted more IL-1ß and expressed more Hsp90 prior to the inhibition of protein clearance, while MCP-1 and IL-6 were reduced at both protein and mRNA levels. Overall, our results suggest that cellular clearance mechanisms might already be dysfunctional, and the inflammasome activated, in cells with a disease origin.
Asunto(s)
Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Células Madre Pluripotentes Inducidas/metabolismo , Inflamasomas/genética , Degeneración Macular/etiología , Epitelio Pigmentado de la Retina/metabolismo , Biomarcadores , Línea Celular , Células Cultivadas , Citocinas/metabolismo , Susceptibilidad a Enfermedades , Perfilación de la Expresión Génica , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Inflamasomas/metabolismo , Mediadores de Inflamación/metabolismo , Degeneración Macular/metabolismo , Epitelio Pigmentado de la Retina/citologíaRESUMEN
Chronic inflammation has been associated with several chronic diseases, such as age-related macular degeneration (AMD). The NLRP3 inflammasome is a central proinflammatory signaling complex that triggers caspase-1 activation leading to the maturation of IL-1ß. We have previously shown that the inhibition of the chaperone protein, Hsp90, prevents NLRP3 activation in human retinal pigment epithelial (RPE) cells; these are cells which play a central role in the pathogenesis of AMD. In that study, we used a well-known Hsp90 inhibitor geldanamycin, but it cannot be used as a therapy due to its adverse effects, including ocular toxicity. Here, we have tested the effects of a novel Hsp90 inhibitor, TAS-116, on NLRP3 activation using geldanamycin as a reference compound. Using our existing protocol, inflammasome activation was induced in IL-1α-primed ARPE-19 cells with the proteasome and autophagy inhibitors MG-132 and bafilomycin A1, respectively. Intracellular caspase-1 activity was determined using a commercial caspase-1 activity kit and the FLICA assay. The levels of IL-1ß were measured from cell culture medium samples by ELISA. Cell viability was monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and lactate dehydrogenase (LDH) measurements. Our findings show that TAS-116 could prevent the activation of caspase-1, subsequently reducing the release of mature IL-1ß. TAS-116 has a better in vitro therapeutic index than geldanamycin. In summary, TAS-116 appears to be a well-tolerated Hsp90 inhibitor, with the capability to prevent the activation of the NLRP3 inflammasome in human RPE cells.
Asunto(s)
Benzamidas/farmacología , Células Epiteliales/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Pirazoles/farmacología , Epitelio Pigmentado de la Retina/patología , Benzoquinonas/farmacología , Caspasa 1/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Lactamas Macrocíclicas/farmacologíaRESUMEN
Retinal pigment epithelial (RPE) cells maintain homeostasis at the retina and they are under continuous oxidative stress. Cigarette smoke is a prominent environmental risk factor for age-related macular degeneration (AMD), which further increases the oxidant load in retinal tissues. In this study, we measured oxidative stress and inflammatory markers upon cigarette smoke-derived hydroquinone exposure on human ARPE-19 cells. In addition, we studied the effects of commercial Resvega product on hydroquinone-induced oxidative stress. Previously, it was observed that Resvega induces autophagy during impaired protein clearance in ARPE-19 cells, for which it has the potential to alleviate pro-inflammatory pathways. Cell viability was determined while using the lactate dehydrogenase (LDH) and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and the cytokine levels were measured using the enzyme-linked immunosorbent assay (ELISA). Reactive oxygen species (ROS) production were measured using the 2',7'-dichlorofluorescin diacetate (H2DCFDA) probe. Hydroquinone compromised the cell viability and increased ROS production in ARPE-19 cells. Resvega significantly improved cell viability upon hydroquinone exposure and reduced the release of interleukin (IL)-8 and monocytic chemoattractant protein (MCP)-1 from RPE cells. Resvega, N-acetyl-cysteine (NAC) and aminopyrrolidine-2,4-dicarboxylic acid (APDC) alleviated hydroquinone-induced ROS production in RPE cells. Collectively, our results indicate that hydroquinone induces cytotoxicity and increases oxidative stress through NADPH oxidase activity in RPE cells, and resveratrol-containing Resvega products prevent those adverse effects.
Asunto(s)
Hidroquinonas/farmacología , Estrés Oxidativo/efectos de los fármacos , Antioxidantes/farmacología , Biomarcadores , Supervivencia Celular/efectos de los fármacos , Citocinas/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
Age-related macular degeneration (AMD) is a complex eye disease in which decline in autophagy leads to the accumulation of sequestosome 1/p62 (SQSTM1/p62)-labeled waste material inside the retinal pigment epithelial (RPE) cells, and the condition results in activation of the inflammasome signaling and IL-1ß secretion. Here, we have studied the role of SQSTM1/p62 in the production of IL-6, IL-8, and MCP-1 in the presence or absence of IL-1ß. SQSTM1/p62 was either overexpressed or silenced in ARPE-19 cells, which were then exposed to IL-1ß. Alternatively, bafilomycin A was used to demonstrate the functional decline of autophagy with increased SQSTM1/p62 levels. The protein concentration of SQSTM1/p62 was measured using the western blot technique, and interleukin levels were determined by ELISA. In IL-1ß-loaded RPE cells, SQSTM1/p62 depletion and overexpression increased the production of MCP-1 and IL-8, respectively. Neither knock-down nor overexpression of SQSTM1/p62 induced the release of IL-6. Our data suggest that SQSTM1/p62 is a significant factor in inflammatory responses, especially following the inflammasome activation.
Asunto(s)
Células Epiteliales/metabolismo , Interleucina-1beta/metabolismo , Degeneración Macular/patología , Epitelio Pigmentado de la Retina/fisiopatología , Proteína Sequestosoma-1/metabolismo , Línea Celular , Quimiocina CCL2/metabolismo , Humanos , Inflamasomas/metabolismo , Interleucina-8/metabolismo , Macrólidos/farmacología , Epitelio Pigmentado de la Retina/citologíaRESUMEN
BACKGROUND/AIMS: Previously, we demonstrated that blockade of the intracellular clearance systems in human retinal pigment epithelial (RPE) cells by MG-132 and bafilomycin A1 (BafA) induces NLRP3 inflammasome signaling. Here, we have explored the activation mechanisms behind this process. NLRP3 is an intracellular receptor detecting factors ranging from the endogenous alarmins and adenosine triphosphate (ATP) to ultraviolet radiation and solid particles. Due to the plethora of triggers, the activation of NLRP3 is often indirect and can be mediated through several alternative pathways. Potassium efflux, lysosomal rupture, and oxidative stress are currently the main mechanisms associated with many activators. METHODS: NLRP3 inflammasomes were activated in human RPE cells by blocking proteasomes and autophagy using MG-132 and bafilomycin A1 (BafA), respectively. P2X7 inhibitor A740003, potassium chloride (KCl), and glyburide, or N-acetyl-L-cysteine (NAC), ammonium pyrrolidinedithiocarbamate (APDC), diphenyleneiodonium chloride (DPI), and mito-TEMPO were added to cell cultures in order to study the role of potassium efflux and oxidative stress, respectively. IL-1ß was measured using the ELISA method. ATP levels and cathepsin B activity were examined using commercial kits, and ROS levels using the fluorescent dye 2´,7´-dichlorodihydrofluorescein diacetate (DCFDA). RESULTS: Elevated extracellular potassium prevented the priming factor IL-1α from inducing the production of reactive oxygen species (ROS). It also prevented IL-1ß release after exposure of primed cells to MG-132 and BafA. Inflammasome activation increased extracellular ATP levels, which did not appear to trigger significant potassium efflux. The activity of the lysosomal enzyme, cathepsin B, was reduced by MG-132 and BafA, suggesting that cathepsin B was not playing any role in this phenomenon. Instead, MG-132 triggered ROS production already 30 min after exposure, but treatment with antioxidants blocking NADPH oxidase and mitochondria-derived ROS significantly prevented IL-1ß release after this activating signal. CONCLUSION: Our data suggest that oxidative stress strongly contributes to the NLRP3 inflammasome activation upon dysfunctional cellular clearance. Clarification of inflammasome activation mechanisms provides novel options for alleviating pathological inflammation present in aggregation diseases, such as age-related macular disease (AMD) and Alzheimer's disease.
Asunto(s)
Autofagia/efectos de los fármacos , Inflamasomas/metabolismo , Leupeptinas/farmacología , Macrólidos/farmacología , Estrés Oxidativo/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Catepsina B/metabolismo , Línea Celular , Humanos , Interleucina-1beta/análisis , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Potasio/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
Age-related macular degeneration (AMD) is a common eye disease among the elderly, which can result in impaired vision and irreversible loss of vision. The majority of patients suffer from the dry (also known as the atrophic) form of the disease, which is completely lacking an effective treatment. In the present study, we evaluated the potential of cis-urocanic acid (cis-UCA) to protect human ARPE-19 cells from cell damage and inflammasome activation induced by UVB light. Urocanic acid is a molecule normally present in human epidermis. Its cis-form has recently been found to alleviate UVB-induced inflammasome activation in human corneal epithelial cells. Here, we observed that cis-UCA is well-tolerated also by human retinal pigment epithelial (RPE) cells at a concentration of 100 µg/ml. Moreover, cis-UCA was cytoprotective and efficiently diminished the levels of mature IL-1ß, IL-18, and cleaved caspase-1 in UVB-irradiated ARPE-19 cells. Interestingly, cis-UCA also reduced DNA damage, whereas its effect against ROS production was negligible. Collectively, cis-UCA protected ARPE-19 cells from UVB-induced phototoxicity and inflammasome activation. This study indicates that due to its beneficial properties of preserving cell viability and preventing inflammation, cis-UCA has potential in drug development of chronic ocular diseases, such as AMD.
RESUMEN
Increased oxidative stress, dysfunctional cellular clearance, and chronic inflammation are associated with age-related macular degeneration (AMD). Prolyl oligopeptidase (PREP) is a serine protease that has numerous cellular functions, including the regulation of oxidative stress, protein aggregation, and inflammation. PREP inhibition by KYP-2047 (4-phenylbutanoyl-L-prolyl1(S)-cyanopyrrolidine) has been associated with clearance of cellular protein aggregates and reduced oxidative stress and inflammation. Here, we studied the effects of KYP-2047 on inflammation, oxidative stress, cell viability, and autophagy in human retinal pigment epithelium (RPE) cells with reduced proteasomal clearance. MG-132-mediated proteasomal inhibition in ARPE-19 cells was used to model declined proteasomal clearance in the RPEs of AMD patients. Cell viability was assessed using LDH and MTT assays. The amounts of reactive oxygen species (ROS) were measured using 2',7'-dichlorofluorescin diacetate (H2DCFDA). ELISA was used to determine the levels of cytokines and activated mitogen-activated protein kinases. The autophagy markers p62/SQSTM1 and LC3 were measured with the western blot method. MG-132 induced LDH leakage and increased ROS production in the ARPE-19 cells, and KYP-2047 reduced MG-132-induced LDH leakage. Production of the proinflammatory cytokine IL-6 was concurrently alleviated by KYP-2047 when compared with cells treated only with MG-132. KYP-2047 had no effect on autophagy in the RPE cells, but the phosphorylation levels of p38 and ERK1/2 were elevated upon KYP-2047 exposure, and the inhibition of p38 prevented the anti-inflammatory actions of KYP-2047. KYP-2047 showed cytoprotective and anti-inflammatory effects on RPE cells suffering from MG-132-induced proteasomal inhibition.
RESUMEN
Age-related macular degeneration (AMD) is a retinal disease leading to impaired vision. Cigarette smoke increases the risk for developing AMD by causing increased reactive oxygen species (ROS) production and damage in the retinal pigment epithelium (RPE). We have previously shown that the cigarette tar component hydroquinone causes oxidative stress in human RPE cells. In the present study, we investigated the propensity of hydroquinone to induce the secretion of interleukin (IL)-1ß and IL-18. The activation of these cytokines is usually regulated by the Nucleotide-binding domain, Leucine-rich repeat, and Pyrin domain 3 (NLRP3) inflammasome. ARPE-19 cells were exposed to hydroquinone, and cell viability was monitored using the lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide salt (MTT) assays. Enzyme-linked immunosorbent assays (ELISAs) were used to measure the levels of proinflammatory cytokines IL-1ß and IL-18 as well as NLRP3, caspase-1, and poly (ADP-ribose) polymerase (PARP). Hydroquinone did not change IL-1ß release but significantly increased the secretion of IL-18. Cytoplasmic NLRP3 levels increased after the hydroquinone treatment of IL-1α-primed RPE cells, but IL-18 was equally released from primed and nonprimed cells. Hydroquinone reduced the intracellular levels of PARP, which were restored by treatment with the ROS scavenger N-acetyl-cysteine (NAC). NAC concurrently reduced the NLRP3 levels but had no effect on IL-18 release. In contrast, the NADPH oxidase inhibitor ammonium pyrrolidinedithiocarbamate (APDC) reduced the release of IL-18 but had no effect on the NLRP3 levels. Collectively, hydroquinone caused DNA damage seen as reduced intracellular PARP levels and induced NLRP3-independent IL-18 secretion in human RPE cells.
Asunto(s)
Daño del ADN , Hidroquinonas/farmacología , Inflamasomas/metabolismo , Interleucina-18/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , HumanosRESUMEN
Purpose: The cornea is continually exposed to highly energetic solar UV-B (280-320 nm). Our aim was to investigate whether UV-B triggers the activation of NLRP3 inflammasomes and the production of IL-1ß and/or IL-18 in human corneal epithelial (HCE) cells. Additionally, we studied the capability of cis-urocanic acid (cis-UCA) to prevent inflammasome activation or alleviate inflammation through other signaling pathways. Methods: HCE-2 cell line and primary HCE cells were primed using lipopolysaccharide or TNF-α. Thereafter, cells were exposed to UV-B before or after the addition of cis-UCA or caspase-1 inhibitor. Caspase-1 activity was measured from cell lysates by an enzymatic assay. IL-1ß, IL-18, IL-6, IL-8, and NLRP3 levels were detected using the ELISA method from cell culture media. Additionally, intracellular NLRP3 levels were determined by the Western blot technique, and cytotoxicity was measured by the LDH assay. Results: UV-B exposure significantly increased caspase-1 activity in TNF-α-primed HCE cells. This result was consistent with the concurrently induced IL-1ß secretion. Both caspase-1 activity and release of IL-1ß were reduced by cis-UCA. Additionally, UV-B stimulated the caspase-1-independent production of IL-18, an effect also reduced by cis-UCA. Cis-UCA decreased the release of IL-6, IL-8, and LDH in a time-dependent manner when administered to HCE-2 cells after UV-B exposure. Conclusions: Our findings demonstrate that UV-B activates inflammasomes in HCE cells. Cis-UCA can prevent the secretion of IL-1ß and IL-18 and therapeutically reduces the levels of IL-6, IL-8, and LDH in UV-B-stressed HCE cells.
Asunto(s)
Epitelio Corneal/efectos de los fármacos , Epitelio Corneal/efectos de la radiación , Inflamasomas/metabolismo , Rayos Ultravioleta , Ácido Urocánico/farmacología , Western Blotting , Caspasa 1/metabolismo , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Epitelio Corneal/metabolismo , Humanos , Inflamación/prevención & control , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Lipopolisacáridos/toxicidad , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Mitochondrial dysfunction has been implicated in a wide variety of degenerative diseases, including age-related macular degeneration. Damage to mitochondria and mitochondrial DNA accumulates with age in the postmitotic retinal pigment epithelium (RPE), which could lead to RPE cell death and trigger disease. One possible mechanism for cells to avoid cell death is mitophagy, the targeted clearance of damaged mitochondria by autophagy. Here, we induced mitochondrial damage in human RPE cells (ARPE-19 and hRPE), using antimycin A, an inhibitor of complex III of the electron transport chain, and investigated cellular viability, mitochondrial structure and function, and autophagy activity. We observed that antimycin A evoked dose-dependent cell death, a rapid loss in mitochondrial membrane potential, and a collapse of oxidative phosphorylation. Mitochondria appeared swollen and there was clear damage to their cristae structure. At the same time, cells were undergoing active autophagy and were sensitive to autophagy inhibition by bafilomycin A1 or chloroquine. These results indicate that mitochondrial dysfunction can cause significant RPE damage and that autophagy is an important survival mechanism for cells suffering from mitochondrial damage.
Asunto(s)
Antimicina A/toxicidad , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Mitocondrias/patología , Epitelio Pigmentado de la Retina/patología , Anciano , Línea Celular , Transporte de Electrón/efectos de los fármacos , Femenino , Glucólisis/efectos de los fármacos , Humanos , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Mitofagia/efectos de los fármacos , Fosforilación Oxidativa/efectos de los fármacos , Fenotipo , Epitelio Pigmentado de la Retina/ultraestructuraRESUMEN
Once activated, the intracellular receptor NLRP3 assembles an inflammasome protein complex that facilitates the caspase-1-mediated maturation of IL-1ß and IL-18. Inactive NLRP3 is guarded by a protein complex containing Hsp90. In response to stress stimuli, Hsp90 is released, and NLRP3 can be activated to promote inflammation. In this study, we blocked Hsp90 with geldanamycin and studied the fate of NLRP3 in human retinal pigment epithelial (RPE) cells. RPE cells play a central role in the development of age-related macular degeneration (AMD), a progressive eye disease causing severe vision loss in the elderly. IL-1α-primed ARPE-19 cells, human embryonal stem cell (hESC)-derived RPE cells, and primary human RPE cells were exposed to MG-132 and bafilomycin A to activate NLRP3 via the inhibition of proteasomes and autophagy, respectively. Additionally, RPE cells were treated with geldanamycin at different time points and the levels of NLRP3 and IL-1ß were determined. Caspase-1 activity was measured using a commercial assay. Geldanamycin prevented the activation of the inflammasome in human RPE cells. NLRP3 released from its protective complex became degraded by autophagy or secreted from the cells. Controlled destruction of NLRP3 is a potential way to regulate the inflammation associated with chronic diseases, such as AMD.
Asunto(s)
Inflamación/genética , Degeneración Macular/genética , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Estrés Fisiológico/genética , Autofagia/efectos de los fármacos , Autofagia/genética , Benzoquinonas/farmacología , Caspasa 1/genética , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Células Madre Embrionarias Humanas/efectos de los fármacos , Células Madre Embrionarias Humanas/metabolismo , Humanos , Inflamasomas/efectos de los fármacos , Inflamasomas/genética , Inflamación/patología , Interleucina-18/genética , Interleucina-1beta/genética , Lactamas Macrocíclicas/farmacología , Macrólidos/farmacología , Degeneración Macular/patología , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/genética , Epitelio Pigmentado de la RetinaRESUMEN
RNA-binding protein dysregulation and altered expression of proteins involved in the autophagy/proteasome pathway play a role in many neurodegenerative disease onset/progression, including age-related macular degeneration (AMD). HuR/ELAVL1 is a master regulator of gene expression in human physiopathology. In ARPE-19 cells exposed to the proteasomal inhibitor MG132, HuR positively affects at posttranscriptional level p62 expression, a stress response gene involved in protein aggregate clearance with a role in AMD. Here, we studied the early effects of the proautophagy AICAR + MG132 cotreatment on the HuR-p62 pathway. We treated ARPE-19 cells with Erk1/2, AMPK, p38MAPK, PKC, and JNK kinase inhibitors in the presence of AICAR + MG132 and evaluated HuR localization/phosphorylation and p62 expression. Two-hour AICAR + MG132 induces both HuR cytoplasmic translocation and threonine phosphorylation via the Erk1/2 pathway. In these conditions, p62 mRNA is loaded on polysomes and its translation in de novo protein is favored. Additionally, for the first time, we report that JNK can phosphorylate HuR, however, without modulating its localization. Our study supports HuR's role as an upstream regulator of p62 expression in ARPE-19 cells, helps to understand better the early events in response to a proautophagy stimulus, and suggests that modulation of the autophagy-regulating kinases as potential therapeutic targets for AMD may be relevant.
Asunto(s)
Proteína 1 Similar a ELAV/metabolismo , Sistema de Señalización de MAP Quinasas , Epitelio Pigmentado de la Retina/metabolismo , Proteína Sequestosoma-1/metabolismo , Autofagia/fisiología , Línea Celular , Humanos , MAP Quinasa Quinasa 4/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/enzimología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Plant-derived polyphenols are known to possess anti-inflammatory and antioxidant effects. In recent years, several studies have investigated their potential benefits for treating chronic diseases associated with prolonged inflammation and excessive oxidative stress, such as age-related macular degeneration (AMD). Previously, two polyphenols, fisetin and luteolin, have been reported to increase the survival of retinal pigment epithelial (RPE) cells suffering from oxidative stress as well as decreasing inflammation but the benefits of polyphenol therapy seem to depend on the model system used. Our aim was to analyze the effects of fisetin and luteolin on inflammation and cellular viability in a model of nonoxidative DNA damage-induced cell death in human RPE (hRPE) cells. Pretreatment of ARPE-19 or primary hRPE cells with the polyphenols augmented etoposide-induced cell death as measured by the lactate dehydrogenase and 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. However, the treatment was able to reduce the release of two proinflammatory cytokines, IL-6 and IL-8, which were determined by enzyme-linked Immunosorbent assay. Analyses of caspase 3 activity, p53 acetylation and SIRT1 protein levels revealed the apoptotic nature of etoposide-evoked cell death and that fisetin and luteolin augmented the etoposide-induced acetylation of p53 and decreased SIRT1 levels. Taken together, our findings suggest that the cytoprotective effects of fisetin and luteolin depend on the stressor they need to combat, whereas their anti-inflammatory potential is sustained over a variety of model systems. Careful consideration of disease pathways will be necessary before fisetin or luteolin can be recommended as therapeutic agents for inflammatory diseases in general and specifically AMD.
Asunto(s)
Daño del ADN/efectos de los fármacos , Flavonoides/farmacología , Luteolina/farmacología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Acetilación/efectos de los fármacos , Antiinflamatorios no Esteroideos/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular , Citocinas/metabolismo , Suplementos Dietéticos , Etopósido/efectos adversos , Flavonoles , Humanos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Retinitis/tratamiento farmacológico , Sirtuina 1/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Porous silicon (PSi) is a promising material for the delivery and sustained release of therapeutic molecules in various tissues. Due to the constant rinsing of cornea by tear solution as well as the short half-life of intravitreal drugs, the eye is an attractive target for controlled drug delivery systems, such as PSi microparticles. Inherent barriers ensure that PSi particles are retained in the eye, releasing drugs at the desired speed until they slowly break down into harmless silicic acid. Here, we have examined the in vitro cytotoxicity of positively and negatively charged thermally oxidized (TOPSi) and thermally carbonized (TCPSi) porous silicon microparticles on human corneal epithelial (HCE) and retinal pigment epithelial (ARPE-19) cells. In addition to ocular assessment under an inverted microscope, cellular viability was evaluated using the CellTiter Blue™, CellTiter Fluor™, and lactate dehydrogenase (LDH) assays. CellTiter Fluor proved to be a suitable assay but due to non-specific and interfering responses, neither CellTiter Blue nor LDH assays should be used when evaluating PSi particles. Our results suggest that the toxicity of PSi particles is concentration-dependent, but at least at concentrations less than 200µg/ml, both positively and negatively charged PSi particles are well tolerated by human corneal and retinal epithelial cells and therefore applicable for delivering drug molecules into ocular tissues.
Asunto(s)
Córnea/efectos de los fármacos , Sistemas de Liberación de Medicamentos/métodos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Silicio/administración & dosificación , Silicio/toxicidad , Administración Oftálmica , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Córnea/fisiología , Relación Dosis-Respuesta a Droga , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/toxicidad , Humanos , Porosidad , Epitelio Pigmentado de la Retina/fisiologíaRESUMEN
Success of the long-term glaucoma therapy and preservation of the visual function strongly depend on patients' compliance which may be affected by the inconvenience of treatment and its side effects. Recently, introduction of preservative-free anti-glaucoma agents has become an important step towards improved glaucoma care by eliminating the negative effects of preservatives on the eye surface. Although, newly developed eye drop formulations do not contain standard preservatives, they still can be harmful to ocular surface due to other excipients. In this study, we compared tolerability of commercial preservative-free (pf) prostaglandin analogues (pf tafluprost, pf latanoprost and pf bimatoprost) in long-term topical application in rabbits in vivo. We found that after eight weeks treatment, pf latanoprost was the worst tolerated among the tested drops. It expressed increased conjunctival redness and blinking frequency. Furthermore, it caused increased LDH release in the aqueous humour, infiltration of macrophages in the eyelids and visible defects in conjunctival goblet cells. However, we did not detect increased levels of inflammatory markers in the tear fluid or in the aqueous humour. Based on our study, we suspect that these negative effects are related to excipients included in pf latanoprost formulation.
Asunto(s)
Ojo/efectos de los fármacos , Ojo/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Prostaglandinas Sintéticas/administración & dosificación , Prostaglandinas Sintéticas/efectos adversos , Administración Tópica , Animales , Biomarcadores/metabolismo , Parpadeo/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Conjuntiva/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Ojo/metabolismo , Párpados/citología , Párpados/efectos de los fármacos , Femenino , L-Lactato Deshidrogenasa/metabolismo , Conservadores Farmacéuticos , Prostaglandinas Sintéticas/química , Conejos , Factores de TiempoRESUMEN
Degeneration of retinal pigment epithelial (RPE) cells is a clinical hallmark of age-related macular degeneration (AMD), the leading cause of blindness among aged people in the Western world. Both inflammation and oxidative stress are known to play vital roles in the development of this disease. Here, we assess the ability of fisetin and luteolin, to protect ARPE-19 cells from oxidative stress-induced cell death and to decrease intracellular inflammation. We also compare the growth and reactivity of human ARPE-19 cells in serum-free and serum-containing conditions. The absence of serum in the culture medium did not prevent ARPE-19 cells from reaching full confluency but caused an increased sensitivity to oxidative stress-induced cell death. Both fisetin and luteolin protected ARPE-19 cells from oxidative stress-induced cell death. They also significantly decreased the release of pro-inflammatory cytokines into the culture medium. The decrease in inflammation was associated with reduced activation of MAPKs and CREB, but was not linked to NF- κB or SIRT1. The ability of fisetin and luteolin to protect and repair stressed RPE cells even after the oxidative insult make them attractive in the search for treatments for AMD.