RESUMEN
BACKGROUND: Ocular toxicity is a severe adverse effect that limits the chronic clinical use of the antiarrhythmic drug amiodarone. Here, we aimed to evaluate the cytoprotective effect of artemisinin and explore the potential signalling pathways in human retinal pigment epithelial (RPE) cell cultures. METHODS: D407 cell cultures were exposed to amiodarone and the impact of artemisinin was evaluated. The key parameters included lactate dehydrogenase (LDH) release, intracellular reactive oxygen species (ROS) generation, and the mitochondrial membrane potential (MMP). We also assessed the protein levels of cleaved caspase-3, cleaved poly (ADP-ribose) polymerase (PARP), phosphorylated adenosine monophosphate-activated protein kinase (AMPK)É (p-AMPK), calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), and nuclear factor erythroid 2-related factor 2 (Nrf2). RESULTS: Artemisinin reduced the cytotoxicity induced by amiodarone, as reflected by decreased LDH release, ROS generation, and MMP disruption. Additionally, artemisinin increased p-AMPK, CaMKK2, and Nrf2 protein levels. Inhibition of AMPK, CaMKK2, or Nrf2 abolished the cytoprotective effect of artemisinin. AMPK activation and Nrf2 knockdown further supported its protective role. CONCLUSIONS: Artemisinin protected RPE cells from amiodarone-induced damage via the CaMKK2/AMPK/Nrf2 pathway. The in vivo experiments in mice confirmed its efficacy in preventing retinal injury caused by amiodarone. These results suggest that an artemisinin-based eye formulation could be repurposed for treating amiodarone-induced ocular toxicity.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Amiodarona , Artemisininas , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina , Citoprotección , Factor 2 Relacionado con NF-E2 , Estrés Oxidativo , Especies Reactivas de Oxígeno , Epitelio Pigmentado de la Retina , Transducción de Señal , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Humanos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/efectos de los fármacos , Citoprotección/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Amiodarona/efectos adversos , Amiodarona/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Artemisininas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Línea Celular , Ratones , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patologíaRESUMEN
BACKGROUND: This study aimed to compare the regressive effects of aflibercept and faricimab on pigment epithelial detachment (PED) in patients with neovascular age-related macular degeneration. METHODS: In total, 41 eyes of 40 patients diagnosed with type 1 macular neovascularization were retrospectively analyzed using multimodal imaging. Of these, 23 eyes were treated with intravitreal aflibercept injections (IVA group), and 18 eyes were treated with intravitreal faricimab (IVFa group), with 3 consecutive injections administered as loading dose therapy. Before treatment and at 1, 2, and 3 months after the first treatment, the maximum height (MH) and maximum diameter (MD) of the PED were measured using optical coherence tomography in each treatment group. RESULTS: In the IVA group, the MH at baseline (215 ± 177 µm) was reduced to 141 ± 150 (P = 0.06), 119 ± 150 (P < 0.01), and 107 ± 150 µm (P < 0.0001) at 1, 2, and 3 months after treatment, respectively. Similarly, in the IVFa group, the MH decreased from 240 ± 195 µm before treatment to 165 ± 170 µm (P = 0.24), 139 ± 142 µm (P < 0.05), and 117 ± 112 µm (P < 0.01) at 1, 2, and 3 months after treatment, respectively. The reduction at 2 and 3 months was significant in both treatments. The mean changes of MH from baseline were -108 ± 142 µm in the IVA group and -124 ± 112 µm in the IVFa group, with no significant difference (P = 0.21). In both groups, the MD did not regress significantly. CONCLUSIONS: The results suggested that the MH of the PED between the IVA and IVFa groups regressed similarly after each loading therapy.
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Inhibidores de la Angiogénesis , Inyecciones Intravítreas , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes de Fusión , Desprendimiento de Retina , Epitelio Pigmentado de la Retina , Tomografía de Coherencia Óptica , Agudeza Visual , Humanos , Receptores de Factores de Crecimiento Endotelial Vascular/uso terapéutico , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/uso terapéutico , Proteínas Recombinantes de Fusión/administración & dosificación , Masculino , Femenino , Estudios Retrospectivos , Desprendimiento de Retina/tratamiento farmacológico , Desprendimiento de Retina/diagnóstico , Anciano , Tomografía de Coherencia Óptica/métodos , Inhibidores de la Angiogénesis/uso terapéutico , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Degeneración Macular Húmeda/tratamiento farmacológico , Degeneración Macular Húmeda/diagnóstico , Degeneración Macular Húmeda/fisiopatología , Anciano de 80 o más Años , Persona de Mediana Edad , Angiografía con Fluoresceína , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
Diabetic retinopathy (DR) is a specific microvascular problem of diabetes, which is mainly caused by hyperglycemia and may lead to rapid vision loss. Dietary polyphenols have been reported to decrease the risk of DR. Apocynum venetum L. leaves are rich in polyphenolic compounds and are popular worldwide for their health benefits as a national tea drink. Building on previous findings of antioxidant activity and aldose reductase inhibition of A. venetum, this study investigated the chemical composition of polyphenol-rich extract of A. venetum leaves (AVL) and its protective mechanism on ARPE-19 cells in hyperglycemia. Ninety-three compounds were identified from AVL by LC-MS/MS, including sixty-eight flavonoids, twenty-one organic acids, and four coumarins. AVL regulated the polyol pathway by decreasing the expression of aldose reductase and the content of sorbitol, enhancing the Na+K+-ATPase activity, and weakening intracellular oxidative stress effectively; it also could regulate the expression of autophagy-related proteins via the AMPK/mTOR/ULK1 signaling pathway to maintain intracellular homeostasis. AVL could restore the polyol pathway, inhibit oxidative stress, and maintain intracellular autophagy to protect cellular morphology and improve DR. The study reveals the phytochemical composition and protective mechanisms of AVL against DR, which could be developed as a functional food and/or candidate pharmaceutical, aiming for retina protection in diabetic retinopathy.
Asunto(s)
Apocynum , Autofagia , Glucosa , Estrés Oxidativo , Extractos Vegetales , Hojas de la Planta , Polifenoles , Epitelio Pigmentado de la Retina , Humanos , Extractos Vegetales/farmacología , Polifenoles/farmacología , Polifenoles/análisis , Hojas de la Planta/química , Autofagia/efectos de los fármacos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Glucosa/metabolismo , Glucosa/efectos adversos , Apocynum/química , Estrés Oxidativo/efectos de los fármacos , Polímeros , Línea Celular , Retinopatía Diabética/prevención & control , Retinopatía Diabética/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Transducción de Señal/efectos de los fármacos , Antioxidantes/farmacología , Aldehído Reductasa/metabolismoRESUMEN
Mitochondrial malfunction, excessive production of reactive oxygen species (ROS), deficient autophagy/mitophagy, and chronic inflammation are hallmarks of age-related macular degeneration (AMD). Metformin has been shown to activate mitophagy, alleviate inflammation, and lower the odds of developing AMD. Here, we explored the ability of metformin to activate mitophagy and alleviate inflammation in retinal pigment epithelium (RPE) cells. Human ARPE-19 cells were pre-treated with metformin for 1 h prior to exposure to antimycin A (10 µM), which induced mitochondrial damage. Cell viability, ROS production, and inflammatory cytokine production were measured, while autophagy/mitophagy proteins were studied using Western blotting and immunocytochemistry. Metformin pre-treatment reduced the levels of proinflammatory cytokines IL-6 and IL-8 to 42% and 65% compared to ARPE-19 cells exposed to antimycin A alone. Metformin reduced the accumulation of the autophagy substrate SQSTM1/p62 (43.9%) and the levels of LC3 I and II (51.6% and 48.6%, respectively) after antimycin A exposure. Metformin also increased the colocalization of LC3 with TOM20 1.5-fold, suggesting active mitophagy. Antimycin A exposure increased the production of mitochondrial ROS (226%), which was reduced by the metformin pre-treatment (84.5%). Collectively, metformin showed anti-inflammatory and antioxidative potential with mitophagy induction in human RPE cells suffering from mitochondrial damage.
Asunto(s)
Inflamación , Metformina , Mitocondrias , Mitofagia , Especies Reactivas de Oxígeno , Epitelio Pigmentado de la Retina , Metformina/farmacología , Humanos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Mitofagia/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Inflamación/patología , Inflamación/tratamiento farmacológico , Especies Reactivas de Oxígeno/metabolismo , Línea Celular , Antimicina A/farmacología , Autofagia/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Degeneración Macular/patología , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/metabolismoRESUMEN
Oxidative damage to human retinal pigment epithelial (RPE) cells is the main cause of age-related macular degeneration (AMD), in our previous work, we showed that ghrelin has an antioxidative effect on human lens epithelium (HLE) cells, however, the studies of using ghrelin in treating the degenerative diseases of the retina have rarely been reported. In this article, we assessed the effect of ghrelin on preventing oxidative stress induced by hydrogen peroxide (H2O2) in ARPE-19 cells and its mechanism. We observed that pretreatment with ghrelin protected ARPE-19 cells from H2O2-induced cell oxidative injuries and apoptosis responses. Furthermore, an oxidative stress-induced mouse model of AMD was established via injection of sodium iodate (NaIO3) to tail veins, and treatment with ghrelin preserved retinal function, and protected photoreceptors.
Asunto(s)
Apoptosis , Modelos Animales de Enfermedad , Ghrelina , Peróxido de Hidrógeno , Degeneración Macular , Estrés Oxidativo , Epitelio Pigmentado de la Retina , Estrés Oxidativo/efectos de los fármacos , Degeneración Macular/etiología , Degeneración Macular/metabolismo , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/prevención & control , Animales , Ghrelina/farmacología , Ghrelina/metabolismo , Humanos , Ratones , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Línea Celular , Apoptosis/efectos de los fármacos , Yodatos , Antioxidantes/farmacología , Ratones Endogámicos C57BL , MasculinoRESUMEN
Glaucine is an aporphine alkaloid with anti-inflammatory, bronchodilator and anti-cancer activities. However, the effects of glaucine in the regulation of age-related macular degeneration (AMD) remain unclear. Herein, we aimed to investigate the anti-angiogenetic and anti-inflammatory effects of glaucine in ARPE-19 cells. ARPE-19 cells were treated with N-(methoxyoxoacetyl)-glycine, methyl ester (DMOG) and cobalt chloride (CoCl2) for induction of hypoxia, while lipopolysaccharide (LPS) treatment was used for elicitation of inflammatory response. Cell viability was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The expression of hypoxia-inducible factor (HIF-1α) and vascular endothelial growth factor (VEGF) were measured by Western blot. The secretion of VEGF, interleukin (IL)-6 and monocyte chemoattractant protein-1 (MCP-1) was detected using enzyme-linked immunosorbent assay (ELISA). Human umbilical vein endothelial cells (HUVECs) were used for tube formation analysis. Expression of HIF-1α and secretion of VEGF were significantly increased under DMOG and CoCl2 induction, whereas glaucine significantly attenuated both HIF-1α expression and VEGF secretion by DMOG- and CoCl2-induced ARPE-19 cells. In addition, glaucine suppressed the tube formation by DMOG- and CoCl2-induced HUVEC cells. Moreover, glaucine also attenuated the production of IL-6 and MCP-1 by LPS-induced ARPE-19 cells. This study indicated that glaucine exhibited anti-angiogenic and anti-inflammatory effects, suggesting that glaucine might have benefits for the treatment of AMD.
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Aporfinas , Supervivencia Celular , Subunidad alfa del Factor 1 Inducible por Hipoxia , Lipopolisacáridos , Epitelio Pigmentado de la Retina , Factor A de Crecimiento Endotelial Vascular , Humanos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/patología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Aporfinas/farmacología , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/patología , Hipoxia de la Célula/efectos de los fármacos , Neovascularización Patológica/tratamiento farmacológico , Antiinflamatorios/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Inhibidores de la Angiogénesis/farmacología , Cobalto/toxicidad , Cobalto/farmacología , Quimiocina CCL2/metabolismo , AngiogénesisRESUMEN
Purpose: Retinal neovascularization is a significant feature of advanced age-related macular degeneration (AMD) and a major cause of blindness in patients with AMD. However, the underlying mechanism of this pathological neovascularization remains unknown. Iron metabolism has been implicated in various biological processes. This study was conducted to investigate the effects of iron metabolism on retinal neovascularization in neovascular AMD (nAMD). Methods: C57BL/6J and very low-density lipoprotein receptor (VLDLR) knockout (Vldlr-/-) mice, a murine model of nAMD, were used in this study. Bulk-RNA sequencing was used to identify differentially expressed genes. Western blot analysis was performed to test the expression of proteins. Iron chelator deferiprone (DFP) was administrated to the mice by oral gavage. Fundus fluorescein angiography was used to evaluate retinal vascular leakage. Immunofluorescence staining was used to detect macrophages and iron-related proteins. Results: RNA sequencing (RNA-seq) results showed altered transferrin expression in the retina and RPE of Vldlr-/- mice. Disrupted iron homeostasis was observed in the retina and RPE of Vldlr-/- mice. DFP mitigated iron overload and significantly reduced retinal neovascularization and vascular leakage. In addition, DFP suppressed the inflammation in Vldlr-/- retinas. The reduced signals of macrophages were observed at sites of neovascularization in the retina and RPE of Vldlr-/- mice after DFP treatment. Further, the IL-6/JAK2/STAT3 signaling pathway was activated in the retina and RPE of Vldlr-/- mice and reversed by DFP treatment. Conclusions: Disrupted iron metabolism may contribute to retinal neovascularization in nAMD. Restoring iron homeostasis by DFP could be a potential therapeutic approach for nAMD.
Asunto(s)
Deferiprona , Modelos Animales de Enfermedad , Homeostasis , Quelantes del Hierro , Hierro , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Retiniana , Animales , Deferiprona/farmacología , Deferiprona/uso terapéutico , Quelantes del Hierro/farmacología , Quelantes del Hierro/uso terapéutico , Ratones , Hierro/metabolismo , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/tratamiento farmacológico , Neovascularización Retiniana/etiología , Neovascularización Retiniana/patología , Angiografía con Fluoresceína , Receptores de LDL/genética , Receptores de LDL/metabolismo , Western Blotting , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/patología , Degeneración Macular Húmeda/tratamiento farmacológico , Degeneración Macular Húmeda/metabolismo , Factor de Transcripción STAT3/metabolismo , MasculinoRESUMEN
Age-related macular degeneration (AMD) and diabetic retinopathy (DR) are common retinal diseases responsible for most blindness in working-age and elderly populations. Oxidative stress and mitochondrial dysfunction play roles in these pathogenesis, and new therapies counteracting these contributors could be of great interest. Some molecules, like coenzyme Q10 (CoQ10), are considered beneficial to maintain mitochondrial homeostasis and contribute to the prevention of cellular apoptosis. We investigated the impact of adding CoQ10 (Q) to a nutritional antioxidant complex (Nutrof Total®; N) on the mitochondrial status and apoptosis in an in vitro hydrogen peroxide (H2O2)-induced oxidative stress model in human retinal pigment epithelium (RPE) cells. H2O2 significantly increased 8-OHdG levels (p < 0.05), caspase-3 (p < 0.0001) and TUNEL intensity (p < 0.01), and RANTES (p < 0.05), caspase-1 (p < 0.05), superoxide (p < 0.05), and DRP-1 (p < 0.05) levels, and also decreased IL1ß, SOD2, and CAT gene expression (p < 0.05) vs. control. Remarkably, Q showed a significant recovery in IL1ß gene expression, TUNEL, TNFα, caspase-1, and JC-1 (p < 0.05) vs. H2O2, and NQ showed a synergist effect in caspase-3 (p < 0.01), TUNEL (p < 0.0001), mtDNA, and DRP-1 (p < 0.05). Our results showed that CoQ10 supplementation is effective in restoring/preventing apoptosis and mitochondrial stress-related damage, suggesting that it could be a valid strategy in degenerative processes such as AMD or DR.
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Apoptosis , Peróxido de Hidrógeno , Estrés Oxidativo , Epitelio Pigmentado de la Retina , Ubiquinona , Humanos , Ubiquinona/análogos & derivados , Ubiquinona/farmacología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Apoptosis/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Antioxidantes/farmacología , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Línea Celular , Suplementos DietéticosRESUMEN
Retinal injury may be exacerbated by iron overload. Astragaloside IV (AS-IV) has potential applications in the food and healthcare industry to promote eye health. We sought to determine the mechanisms responsible for the protective effects of AS-IV on photoreceptor and retinal pigment epithelium cell death induced by iron overload. We conducted in vitro and in vivo experiments involving AS-IV pretreatment. We tested AS-IV for its ability to protect iron-overload mice from retinal injury. In particular, we analyzed the effects of AS-IV on iron overload-induced ferroptosis in 661W and ARPE-19 cells. AS-IV not only attenuated iron deposition and retinal injury in iron-overload mice but also effectively reduced iron overload-induced ferroptotic cell death in 661W and ARPE-19 cells. AS-IV effectively prevented ferroptosis by inhibiting iron accumulation and lipid peroxidation. In addition, inhibiting nuclear factor erythroid 2-related factor 2 (Nrf2) eliminated the protective effect of AS-IV against ferroptosis. The results suggest that ferroptosis might be a significant cause of retinal cell death associated with iron overload. AS-IV provides protection from iron overload-induced ferroptosis, partly by activating the Nrf2 signaling pathway.
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Ferroptosis , Sobrecarga de Hierro , Ratones Endogámicos C57BL , Epitelio Pigmentado de la Retina , Saponinas , Triterpenos , Ferroptosis/efectos de los fármacos , Animales , Triterpenos/farmacología , Triterpenos/uso terapéutico , Saponinas/farmacología , Sobrecarga de Hierro/metabolismo , Sobrecarga de Hierro/tratamiento farmacológico , Ratones , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Modelos Animales de Enfermedad , Peroxidación de Lípido/efectos de los fármacos , Humanos , Enfermedades de la Retina/prevención & control , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/patología , Enfermedades de la Retina/tratamiento farmacológico , Factor 2 Relacionado con NF-E2/metabolismo , Western Blotting , Masculino , Hierro/metabolismoRESUMEN
Primary cilia are membrane-covered microtubule-based structures that protrude from the cell surface and are critical for cell signaling and homeostasis during human development and adulthood. Dysregulation of cilia formation, length, and function can lead to a spectrum of human diseases and syndromes known as ciliopathies. Although some genetic and chemical screens have been performed to define important factors that modulate cilia biogenesis and length control, there are currently no clinical treatments that restore cilia length in patients. We report that the microtubule-targeting agent MI-181(mitotic inhibitor-181) is a potent modulator of cilia length and biogenesis. Treatment of retinal pigment epithelial-1 cells with MI-181 induced an increase in the average size of cilia and in the percent ciliated cells under nonstarved conditions. Importantly, MI-181 was effective at rescuing cilia length and ciliation defects in cells that had been treated with the intraflagellar transport inhibitor Ciliobrevin D or the O-GlcNAc transferase inhibitor OSMI-1. Most importantly, MI-181 induced an increase in cilia length and restored ciliation in cells with compromised shortened cilia at low nanomolar concentrations and did not show an inhibitory response at high concentrations. Therefore, MI-181 represents a lead molecule for developing drugs targeting ciliopathies characterized by shortened cilia.
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Cilios , Humanos , Línea Celular , Cilios/efectos de los fármacos , Cilios/metabolismo , Ciliopatías/metabolismo , Ciliopatías/patología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
Deferasirox (DFS) is an oral iron chelator that is employed in retinal ailments as a neuroprotectant against retinal injury and thus has utility in treating disorders such as excitoneurotoxicity and age-related macular degeneration (AMD). However, the conventional oral route of administration can present several disadvantages, e.g., the need for more frequent dosing and the first-pass effect. Microneedles (MNs) are minimally invasive systems that can be employed for intrascleral drug delivery without pain and can advantageously replace intravitreal injections therapy (IVT) as well as conventional oral routes of delivery for DFS. In this study, DFS was formulated into a nanosuspension (NS) through wet media milling employing PVA as a stabilizer, which was successfully loaded into polymeric dissolving MNs. DFS exhibited a 4-fold increase in solubility in DFS-NS compared to that of pure DFS. Moreover, the DFS-NSs exhibited excellent short-term stability and enhanced thermal stability, as confirmed through thermogravimetric analysis (TGA) studies. The mechanical characterization of the DFS-NS loaded ocular microneedles (DFS-NS-OcMNs), revealed that the system was sufficiently strong for effective scleral penetration. Optical coherence tomography (OCT) images confirmed the insertion of 81.23 ± 7.35 % of the total height of the MN arrays into full-thickness porcine sclera. Scleral deposition studies revealed 64 % drug deposition after just 5 min of insertion from DFS-NS-loaded ocular microneedles (OcMNs), which was almost 5 times greater than the deposition from pure DFS-OcMNs. Furthermore, both DFS and DFS-NS-OcMN exhibited remarkable cell viability when evaluated on human retinal pigment (ARPE) cells, suggesting their safety and appropriateness for use in the human eye. Therefore, loading DFS-NS into novel MN devices is a promising technique for effectively delivering DFS to the posterior segment of the eye in a minimally invasive manner.
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Deferasirox , Sistemas de Liberación de Medicamentos , Quelantes del Hierro , Agujas , Deferasirox/administración & dosificación , Deferasirox/farmacocinética , Animales , Porcinos , Quelantes del Hierro/administración & dosificación , Solubilidad , Suspensiones , Esclerótica/metabolismo , Humanos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Nanopartículas/administración & dosificación , Supervivencia Celular/efectos de los fármacos , Línea Celular , Administración Oftálmica , Microinyecciones/métodos , Estabilidad de Medicamentos , Tomografía de Coherencia ÓpticaRESUMEN
Malattia Leventinese/Doyne honeycomb retinal dystrophy (ML/DHRD) is an age-related macular degeneration-like (AMD-like) retinal dystrophy caused by an autosomal dominant R345W mutation in the secreted glycoprotein, fibulin-3 (F3). To identify new small molecules that reduce F3 production in retinal pigmented epithelium (RPE) cells, we knocked-in a luminescent peptide tag (HiBiT) into the endogenous F3 locus that enabled simple, sensitive, and high-throughput detection of the protein. The GSK3 inhibitor, CHIR99021 (CHIR), significantly reduced F3 burden (expression, secretion, and intracellular levels) in immortalized RPE and non-RPE cells. Low-level, long-term CHIR treatment promoted remodeling of the RPE extracellular matrix, reducing sub-RPE deposit-associated proteins (e.g., amelotin, complement component 3, collagen IV, and fibronectin), while increasing RPE differentiation factors (e.g., tyrosinase, and pigment epithelium-derived factor). In vivo, treatment of 8-month-old R345W+/+ knockin mice with CHIR (25 mg/kg i.p., 1 mo) was well tolerated and significantly reduced R345W F3-associated AMD-like basal laminar deposit number and size, thereby preventing the main pathological feature in these mice. This is an important demonstration of small molecule-based prevention of AMD-like pathology in ML/DHRD mice and may herald a rejuvenation of interest in GSK3 inhibition for the treatment of retinal degenerative diseases, including potentially AMD itself.
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Proteínas de la Matriz Extracelular , Matriz Extracelular , Degeneración Macular , Epitelio Pigmentado de la Retina , Animales , Ratones , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Degeneración Macular/patología , Degeneración Macular/genética , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/metabolismo , Humanos , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/efectos de los fármacos , Piridinas/farmacología , Pirimidinas/farmacología , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/genética , Modelos Animales de Enfermedad , Distrofias Retinianas/metabolismo , Distrofias Retinianas/patología , Distrofias Retinianas/genética , Drusas del Disco Óptico/congénitoRESUMEN
The aim of the present study was to elucidate unknown effects of intraocular fatty acids (ioFAs) including palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), arachidonic acid (C20:4), eicosapentaenoic acid (EPA, C20:5) and docosahexaenoic acid (DHA, C22:6) on the outer blood-retinal barrier (oBRB). For this purpose, human retinal pigment epithelium cell line ARPE19 was subjected to analyses for evaluating the following biological phenotypes: (1) cell viability, (2) cellular metabolic functions, (3) barrier functions by trans-epithelial electrical resistance (TEER), and (4) expression of tight junction (TJ) molecules. In the presence of 100 nM ioFAs, no significant effects on cell viability of ARPE19 cells was observed. While treatment with EPA or DHA tended to reduce non-mitochondrial oxygen consumption, most indices in mitochondrial functions were not markedly affected by treatment with ioFAs in ARPE19 cells. On the other hand, ioFAs except for palmitic acid and stearic acid significantly increased basal extracellular acidification rates, suggesting activated glycolysis or increased lactate production. Interestingly, TEER values of planar ARPE19 monolayer were significantly increased by treatment any ioFAs. Consistently, gene expression levels of TJ proteins were increased by treatment with ioFAs. Collectively, the findings presented herein suggest that ioFAs may contribute to reinforcement of barrier functions of the oBRB albeit there are some differences in biological effects depending on the type of ioFAs.
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Barrera Hematorretinal , Epitelio Pigmentado de la Retina , Humanos , Barrera Hematorretinal/metabolismo , Barrera Hematorretinal/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/citología , Línea Celular , Ácidos Grasos/metabolismo , Ácidos Grasos/farmacología , Supervivencia Celular/efectos de los fármacos , Ácido Palmítico/farmacología , Ácidos Docosahexaenoicos/farmacología , Ácidos Esteáricos/farmacología , Ácido Linoleico/farmacología , Ácido Eicosapentaenoico/farmacología , Ácido Oléico/farmacología , Uniones Estrechas/metabolismo , Uniones Estrechas/efectos de los fármacos , Ácido Araquidónico/farmacología , Ácido Araquidónico/metabolismoRESUMEN
Physiologically relevant in vitro models of the human outer retina are required to better elucidate the complex interplay of retinal tissue layers and investigate their role in retinal degenerative disorders. Materials currently used to mimic the function of Bruch's membrane fail to replicate a range of important structural, mechanical, and biochemical properties. Here, we detail the fabrication of a surface-functionalized, fibrous collagen I membrane. We demonstrate its ability to better replicate a range of important material properties akin to the function of human Bruch's membrane when compared with a commonly utilized synthetic polyethylene terephthalate alternative. We further reveal the ability of this membrane to support the culture of the ARPE-19 cell line, as well as human pluripotent stem cell-derived RPE-like cells and human umbilical vein endothelial cells. This material could provide greater physiological relevance to the native Bruch's membrane than current synthetic materials and further improve the outcomes of in vitro outer retinal models.
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Lámina Basal de la Coroides , Colágeno Tipo I , Retina , Humanos , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología , Lámina Basal de la Coroides/metabolismo , Lámina Basal de la Coroides/química , Línea Celular , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Tereftalatos Polietilenos/química , Retina/citología , Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacosRESUMEN
Inflammation-induced choroidal neovascularization followed by the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPEs) is a cause of neovascular age-related macular degeneration (nAMD). RPE-derived myofibroblasts overproduce extracellular matrix, leading to subretinal fibrosis. We already have demonstrated that benzylphenylurea (BPU) derivatives inhibit the function of cancer-associated fibroblasts. Here, we investigated the anti-myofibroblast effects of BPU derivatives and examined such BPU activity on subretinal fibrosis. A BPU derivative, BPU17, exhibits the most potent anti-myofibroblast activity among dozens of BPU derivatives and inhibits subretinal fibrosis in a mouse model of retinal degeneration. Investigations with primary cultured RPEs reveal that BPU17 suppresses cell motility and collagen synthesis in RPE-derived myofibroblasts. These effects depend on repressing the serum response factor (SRF)/CArG-box-dependent transcription. BPU17 inhibits the expression of SRF cofactor, cysteine and glycine-rich protein 2 (CRP2), which activates the SRF function. Proteomics analysis reveals that BPU17 binds to prohibitin 1 (PHB1) and inhibits the PHB1-PHB2 interaction, resulting in mild defects in mitochondrial function. This impairment causes a decrease in the expression of CRP2 and suppresses collagen synthesis. Our findings suggest that BPU17 is a promising agent against nAMD and the close relationship between PHB function and EMT.
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Fibrosis , Miofibroblastos , Prohibitinas , Proteínas Represoras , Animales , Proteínas Represoras/metabolismo , Humanos , Ratones , Miofibroblastos/efectos de los fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patología , Fibrosis/tratamiento farmacológico , Antifibróticos/farmacología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Movimiento Celular/efectos de los fármacos , Ratones Endogámicos C57BL , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/metabolismo , Degeneración Macular/patología , Células Cultivadas , Factor de Respuesta Sérica/metabolismo , Factor de Respuesta Sérica/antagonistas & inhibidoresRESUMEN
PURPOSE: The abnormal growth factors-induced epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells was known as a vital pathogenesis of proliferative vitreoretinopathy (PVR). This study aims to explore how survivin inhibition affects EMT induced by epidermal growth factor (EGF) in RPE cells. METHODS: Human primary RPE cells were identified in vitro. EMT in RPE cells was induced by EGF. Inhibition of survivin in RPE cells was accomplished through the use of a survivin inhibitor (YM155) and survivin siRNA. The viability, proliferation and migration of RPE cells was detected by methylthiazol tetrazolium assay, bromodeoxyuridine labeling assay, and wound healing assay, respectively. The EGF receptor /mitogen-activated protein kinase (EGFR/MAPK) proteins and EMT-related proteins were measured by western blot and immunofluorescence assay. RESULTS: EGF induced significant EMT in RPE cells, activated the phosphorylation of EGFR/MAPK signaling proteins, and caused changes to EMT-related proteins. YM155 suppressed RPE cells' viability, proliferation, and migration; induced the phosphorylation of EGFR, JNK, and P38MAPK; and down regulated EGFR and phosphorylated ERK. YM155 also increased expression of E-cadherin and ZO-1 proteins and reduced expression of N-cadherin, Vimentin, and α-SMA proteins. The EGF-induced increase of RPE cell proliferation and migration was constrained by survivin inhibition. Moreover, survivin inhibition in RPE cells suppressed the EGF-caused phosphorylation of EGFR/MAPK proteins and attenuated the EGF-induced reduction of E-cadherin and ZO-1 proteins and increase of N-cadherin, Vimentin, and α-SMA proteins. CONCLUSIONS: Survivin inhibition attenuates EGF-induced EMT of RPE cells by affecting the EGFR/MAPK signaling pathway. Survivin might be a promising target for preventing PVR.
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Cadherinas , Movimiento Celular , Proliferación Celular , Factor de Crecimiento Epidérmico , Transición Epitelial-Mesenquimal , Receptores ErbB , Imidazoles , Sistema de Señalización de MAP Quinasas , Naftoquinonas , Epitelio Pigmentado de la Retina , Survivin , Humanos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Survivin/metabolismo , Survivin/antagonistas & inhibidores , Receptores ErbB/metabolismo , Receptores ErbB/antagonistas & inhibidores , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Naftoquinonas/farmacología , Proliferación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Imidazoles/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Cadherinas/metabolismo , Supervivencia Celular/efectos de los fármacos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Fosforilación/efectos de los fármacos , Vimentina/metabolismo , Proteína de la Zonula Occludens-1/metabolismo , Células Cultivadas , Actinas/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
BACKGROUND: Age-related macular degeneration (AMD) is a prevalent ocular pathology affecting mostly the elderly population. AMD is characterized by a progressive retinal pigment epithelial (RPE) cell degeneration, mainly caused by an impaired antioxidative defense. One of the AMD therapeutic procedures involves injecting healthy RPE cells into the subretinal space, necessitating pure, healthy RPE cell suspensions. This study aims to electrically characterize RPE cells to demonstrate a possibility using simulations to separate healthy RPE cells from a mixture of healthy/oxidized cells by dielectrophoresis. METHODS: BPEI-1 rat RPE cells were exposed to hydrogen peroxide to create an in-vitro AMD cellular model. Cell viability was evaluated using various methods, including microscopic imaging, impedance-based real-time cell analysis, and the MTS assay. Healthy and oxidized cells were characterized by recording their dielectrophoretic spectra, and electric cell parameters (crossover frequency, membrane conductivity and permittivity, and cytoplasm conductivity) were computed. A COMSOL simulation was performed on a theoretical microfluidic-based dielectrophoretic separation chip using these parameters. RESULTS: Increasing the hydrogen peroxide concentration shifted the first crossover frequency toward lower values, and the cell membrane permittivity progressively increased. These changes were attributed to progressive membrane peroxidation, as they were diminished when measured on cells treated with the antioxidant N-acetylcysteine. The changes in the crossover frequency were sufficient for the efficient separation of healthy cells, as demonstrated by simulations. CONCLUSIONS: The study demonstrates that dielectrophoresis can be used to separate healthy RPE cells from oxidized ones based on their electrical properties. This method could be a viable approach for obtaining pure, healthy RPE cell suspensions for AMD therapeutic procedures.
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Supervivencia Celular , Peróxido de Hidrógeno , Degeneración Macular , Epitelio Pigmentado de la Retina , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Animales , Ratas , Peróxido de Hidrógeno/toxicidad , Peróxido de Hidrógeno/farmacología , Electroforesis/métodos , Estrés Oxidativo , Células CultivadasRESUMEN
Cell adhesion to the extracellular matrix and its natural outcome of cell spreading, along with the maintenance of barrier activity, are essential behaviors of epithelial cells, including retinal pigment epithelium (RPE). Disruptions in these characteristics can result in severe vision-threatening diseases such as diabetic macular edema and age-related macular degeneration. However, the precise mechanisms underlying how RPE cells regulate their barrier integrity and cell spreading are not fully understood. This study aims to elucidate the relative importance of upper glycolytic components in governing these cellular behaviors of RPE cells. Electric Cell-Substrate Impedance Sensing (ECIS) technology was utilized to assess in real-time the effects of targeting various upper glycolytic enzymes on RPE barrier function and cell spreading by measuring cell resistance and capacitance, respectively. Specific inhibitors used included WZB117 for Glut1 inhibition, Lonidamine for Hexokinase inhibition, PFK158 for PFKFB3/PFK axis inhibition, and TDZD-8 for Aldolase inhibition. Additionally, the viability of RPE cells was evaluated using a lactate dehydrogenase (LDH) cytotoxicity assay. The most significant decrease in electrical resistance and increase in capacitance of RPE cells were observed due to dose-dependent inhibition of Glut1 using WZB117, as well as Aldolase inhibition with TDZD-8. LDH level analysis at 24-72 h post-treatment with WZB117 (1 and 10 µM) or TDZD-8 (1 µM) showed no significant difference compared to the control, indicating that the disruption of RPE functionality was not attributed to cell death. Lastly, inhibition of other upper glycolytic components, including PFKFB3/PFK with PFK158 or Hexokinase with Lonidamine, did not significantly affect RPE cell behavior. This study provides insights into the varied roles of upper glycolytic components in regulating the functionality of RPE cells. Specifically, it highlights the critical roles of Glut1 and Aldolase in preserving barrier integrity and promoting RPE cell adhesion and spreading. Such understanding will guide the development of safe interventions to treat RPE cell dysfunction in various retinal disorders.
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Glucólisis , Epitelio Pigmentado de la Retina , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/citología , Glucólisis/efectos de los fármacos , Humanos , Transportador de Glucosa de Tipo 1/metabolismo , Hexoquinasa/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Impedancia Eléctrica , Fosfofructoquinasa-2/metabolismo , Fosfofructoquinasa-2/antagonistas & inhibidoresRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Cordyceps cicadae (C.cicadae), named "Chan Hua", an anamorph of Isaria cicadae Miquel, is an entomogenous complex formed by fungi parasitizing on the larvae of cicadas and belongs to the Claviciptaceae family and the genus Codyceps, which traditionally holds a significant place in Chinese ethnopharmacology, specifically for eye clarity and as a remedy for age-related ocular conditions. The underlying mechanisms contributing to its eyesight enhancement and potential effectiveness against Age-related macular degeneration (AMD) remain unexplored. AIM OF THE STUDY: This study aims to elucidate the protective role of C.cicadae and its active ingredient, Myriocin (Myr), against AMD. MATERIALS AND METHODS: A chemical inducer was employed to make retinal pigment epithelium (RPE) damage in vitro and in vivo. The key ingredients of C.cicadae and their related mechanisms for anti-AMD were studied through bioinformatic analysis and molecular biological approaches. RESULTS: Myr was identified through high-performance liquid chromatography (HPLC) as an active ingredient in C.cicadae, and demonstrated a protective effect on RPE cells, reducing the structural damage and cell death induced by sodium iodate (SI). Further, Myr reduced eyelid secretions in AMD mice and restored their retinal structure and function. The differentially expressed genes (DEGs) in Myr treatment are primarily associated with TNF and Necroptosis signaling pathways. Molecular docking indicated a strong affinity between TNF and Myr. Myr inhibited the TNF signaling pathway thereby reducing the expression of inflammatory factors in ARPE-19 cells. Additionally, Myr had consistent action with the necroptosis inhibitor Necrostatin-1 (Nec-1), inhibited the RIPK1/RIPK3/MLKL pathway thereby protecting ARPE-19 cells. CONCLUSION: The findings present Myr, as a potent protector against SI-induced AMD, predominantly through modulation of the TNF-RIPK1/RIPK3/MLKL signaling pathway, offering the insights of therapeutic C.cicadae as viable candidates for AMD treatment.
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Cordyceps , Yodatos , Degeneración Macular , Epitelio Pigmentado de la Retina , Factor de Necrosis Tumoral alfa , Animales , Degeneración Macular/tratamiento farmacológico , Cordyceps/química , Ratones , Factor de Necrosis Tumoral alfa/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Transducción de Señal/efectos de los fármacos , Humanos , Línea Celular , Ratones Endogámicos C57BL , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Masculino , Necroptosis/efectos de los fármacos , Ácidos Grasos MonoinsaturadosRESUMEN
Current diabetic retinopathy (DR) treatment involves blood glucose regulation combined with laser photocoagulation or intravitreal injection of vascular endothelial growth factor (VEGF) antibodies. However, due to the complex pathogenesis and cross-interference of multiple biochemical pathways, these interventions cannot block disease progression. Recognizing the critical role of the retinal microenvironment (RME) in DR, it is hypothesized that reshaping the RME by simultaneously inhibiting primary and secondary blood-retinal barrier (BRB) injury can attenuate DR. For this, a glucose-responsive hydrogel named Cu-PEI/siMyD88@GEMA-Con A (CSGC) is developed that effectively delivers Cu-PEI/siMyD88 nanoparticles (NPs) to the retinal pigment epithelium (RPE). The Cu-PEI NPs act as antioxidant enzymes, scavenging ROS and inhibiting RPE pyroptosis, ultimately blocking primary BRB injury by reducing microglial activation and Th1 differentiation. Simultaneously, MyD88 expression silence in combination with the Cu-PEI NPs decreases IL-18 production, synergistically reduces VEGF levels, and enhances tight junction proteins expression, thus blocking secondary BRB injury. In summary, via remodeling the RME, the CSGC hydrogel has the potential to disrupt the detrimental cycle of cross-interference between primary and secondary BRB injury, providing a promising therapeutic strategy for DR.