RESUMEN
Monocyte activation plays an important role in diabetic complications such as diabetic retinopathy (DR). However, the regulation of monocyte activation in diabetes remains elusive. Fenofibrate, an agonist of peroxisome proliferator-activated receptor-α (PPARα), has shown robust therapeutic effects on DR in patients with type 2 diabetes. Here we found that PPARα levels were significantly downregulated in monocytes from patients with diabetes and animal models, correlating with monocyte activation. Fenofibrate attenuated monocyte activation in diabetes, while PPARα knockout alone induced monocyte activation. Furthermore, monocyte-specific PPARα overexpression ameliorated, while monocyte-specific PPARα knockout aggravated monocyte activation in diabetes. PPARα knockout impaired mitochondrial function while also increasing glycolysis in monocytes. PPARα knockout increased cytosolic mitochondrial DNA release and activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway in monocytes under diabetic conditions. STING knockout or STING inhibitor attenuated monocyte activation induced by diabetes or by PPARα knockout. These observations suggest that PPARα negatively regulates monocyte activation through metabolic reprogramming and interaction with the cGAS-STING pathway.
Asunto(s)
Diabetes Mellitus Tipo 2 , Retinopatía Diabética , Fenofibrato , Animales , PPAR alfa/genética , PPAR alfa/metabolismo , Fenofibrato/farmacología , Fenofibrato/uso terapéutico , Monocitos/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Retinopatía Diabética/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismoRESUMEN
Microglial activation and subsequent pathological neuroinflammation contribute to diabetic retinopathy (DR). However, the underlying mechanisms of microgliosis, and means to effectively suppress pathological microgliosis, remain incompletely understood. Peroxisome proliferator-activated receptor alpha (PPARα) is a transcription factor that regulates lipid metabolism. The present study aimed to determine if PPARα affects pathological microgliosis in DR. In global Pparα mice, retinal microglia exhibited decreased structural complexity and enlarged cell bodies, suggesting microglial activation. Microglia-specific conditional Pparα-/- (PCKO) mice showed decreased retinal thickness as revealed by optical coherence tomography. Under streptozotocin (STZ)-induced diabetes, diabetic PCKO mice exhibited decreased electroretinography response, while diabetes-induced retinal dysfunction was alleviated in diabetic microglia-specific Pparα-transgenic (PCTG) mice. Additionally, diabetes-induced retinal pericyte loss was exacerbated in diabetic PCKO mice and alleviated in diabetic PCTG mice. In cultured microglial cells with the diabetic stressor 4-HNE, metabolic flux analysis demonstrated that Pparα ablation caused a metabolic shift from oxidative phosphorylation to glycolysis. Pparα deficiency also increased microglial STING and TNF-α expression. Taken together, these findings revealed a critical role for PPARα in pathological microgliosis, neurodegeneration, and vascular damage in DR, providing insight into the underlying molecular mechanisms of microgliosis in this context and suggesting microglial PPARα as a potential therapeutic target.
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Diabetes Mellitus Experimental , Retinopatía Diabética , PPAR alfa , Animales , Ratones , Diabetes Mellitus Experimental/metabolismo , Retinopatía Diabética/patología , Microglía/metabolismo , PPAR alfa/metabolismo , Retina/metabolismoRESUMEN
Macular edema caused by retinal vascular leakage and ocular neovascularization are the leading causes of severe vision loss in diabetic retinopathy (DR) and age-related macular degeneration (AMD) patients. Oral administration of fenofibrate, a PPARα agonist, has shown therapeutic effects on macular edema and retinal neovascularization in diabetic patients. To improve the drug delivery to the retina and its efficacy, we have developed a nano-emulsion-based fenofibrate eye drop formulation that delivered significantly higher amounts of the drug to the retina compared to the systemic administration, as measured by liquid chromatography-mass spectrometer (LC-MS). The fenofibrate eye drop decreased leukocytes adherent to retinal vasculature and attenuated overexpression of multiple inflammatory factors in the retina of very low-density lipoprotein receptor knockout (Vldlr-/-) mice, a model manifesting AMD phenotypes, and streptozotocin-induced diabetic rats. The fenofibrate eye drop also reduced retinal vascular leakage in these models. The laser-induced choroidal neovascularization was also alleviated by the fenofibrate eye drop. There were no detectable ocular toxicities associated with the fenofibrate eye drop treatment. These findings suggest that fenofibrate can be delivered efficiently to the retina through topical administration of the nano-emulsion eye drop, which has therapeutic potential for macular edema and neovascularization.
RESUMEN
Patients with diabetes often experience visual defects before any retinal pathologies are detected. The molecular mechanism for the visual defects in early diabetes has not been elucidated. Our previous study reported that in early diabetic retinopathy (DR), rhodopsin levels were reduced due to impaired 11-cis-retinal regeneration. Interphotoreceptor retinol-binding protein (IRBP) is a visual cycle protein and important for 11-cis-retinal generation. IRBP levels are decreased in the vitreous and retina of DR patients and animal models. To determine the role of IRBP downregulation in the visual defects in early DR, we induced diabetes in transgenic mice overexpressing IRBP in the retina. IRBP overexpression prevented diabetes-induced decline of retinal function. Furthermore, IRBP overexpression also prevented decreases of rhodopsin levels and 11-cis-retinal generation in diabetic mice. Diabetic IRBP transgenic mice also showed ameliorated retinal oxidative stress, inflammation, apoptosis, and retinal degeneration compared with diabetic wild-type mice. These findings suggest that diabetes-induced IRBP downregulation impairs the regeneration of 11-cis-retinal and rhodopsin, leading to retinal dysfunction in early DR. Furthermore, increased 11-cis-retinal-free opsin constitutively activates the phototransduction pathway, leading to increased oxidative stress and retinal neurodegeneration. Therefore, restored IRBP expression in the diabetic retina may confer a protective effect against retinal degeneration in DR.
Asunto(s)
Retinopatía Diabética/metabolismo , Proteínas del Ojo/metabolismo , Proteínas de Unión al Retinol/metabolismo , Rodopsina/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Western Blotting , Retinopatía Diabética/genética , Proteínas del Ojo/genética , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Inflamación/genética , Inflamación/metabolismo , Ratones , Ratones Transgénicos , Estrés Oxidativo/genética , Estrés Oxidativo/fisiología , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Proteínas de Unión al Retinol/genética , Rodopsina/genética , Tomografía de Coherencia ÓpticaRESUMEN
PURPOSE: Elevated blood levels of C-reactive protein (CRP) are associated with both type 1 and type 2 diabetes and diabetic complications, such as diabetic retinopathy (DR). However, its pathogenic role in DR remains unknown. The present study aims to investigate the potential role of CRP in DR pathogenesis and explore its underlying mechanism. MATERIALS AND METHODS: Human CRP transgenic (hCRP-Tg) rats were employed for streptozotocin (STZ)-induced diabetic and oxygen-induced retinopathy (OIR) models. The retina function was monitored by electroretinography (ERG) and retinal thickness was measured by optical coherence tomography (OCT). TUNEL and cell death ELISA were performed to measure the apoptosis. Oxidative stress was detected by the measurement of reactive oxygen species (ROS) in cells and 3-Nitrotyrosine staining in tissue sections. RESULTS: In non-diabetic condition, hCRP-Tg with elevated hCRP levels in the retinas demonstrated declined ERG responses and decreased retinal thickness. In STZ-induced diabetic condition, overexpression of hCRP deteriorated retinal neurodegeneration as shown by ERG and apoptosis assays. hCRP also exacerbated retinal leukostasis and acellular capillary formation induced by diabetes. In the OIR model, overexpression of hCRP exacerbated retinal neovascularization (NV). In retinal cell lines, hCRP treatment induced cell death and over-production of ROS. Furthermore, hCRP-induced overexpression of pro-inflammatory, pro-oxidative, and pro-angiogenic factors was associated with up-regulation of CD32 and the NF-κB signaling in the retinas. CONCLUSIONS: Elevated hCRP levels play a pathogenic role in DR. Targeting the hCRP-CD32-NF-κB pathway may represent a novel therapeutic strategy for DR.
Asunto(s)
Proteína C-Reactiva/metabolismo , Retinopatía Diabética/metabolismo , Animales , Animales Modificados Genéticamente , Apoptosis , Proteína C-Reactiva/genética , Retinopatía Diabética/complicaciones , Retinopatía Diabética/genética , Retinopatía Diabética/patología , Humanos , Estrés Oxidativo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Retina/metabolismo , Retina/patología , Neovascularización Retiniana/etiología , Neovascularización Retiniana/genética , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/patología , Transducción de SeñalRESUMEN
The purpose of this study was to investigate the protective role of peroxisome proliferator-activated receptor α (PPARα) against diabetic keratopathy and corneal neuropathy. Corneal samples were obtained from human donors with and without diabetes. Streptozotocin-induced diabetic rats and mice were orally treated with PPARα agonist fenofibrate. As shown by immunohistochemistry and Western blotting, PPARα was downregulated in the corneas of humans with diabetes and diabetic rats. Immunostaining of ß-III tubulin demonstrated that corneal nerve fiber metrics were decreased significantly in diabetic rats and mice, which were partially prevented by fenofibrate treatment. As evaluated using a Cochet-Bonnet aesthesiometer, corneal sensitivity was significantly decreased in diabetic mice, which was prevented by fenofibrate. PPARα -/- mice displayed progressive decreases in the corneal nerve fiber density. Consistently, corneal sensitivity was decreased in PPARα -/- mice relative to wild-type mice by 21 months of age. Diabetic mice showed increased incidence of spontaneous corneal epithelial lesion, which was prevented by fenofibrate while exacerbated by PPARα knockout. Western blot analysis revealed significantly altered neurotrophic factor levels in diabetic rat corneas, which were partially restored by fenofibrate treatment. These results indicate that PPARα protects the corneal nerve from degeneration induced by diabetes, and PPARα agonists have therapeutic potential in the treatment of diabetic keratopathy.
Asunto(s)
Córnea/inervación , Diabetes Mellitus Experimental/patología , Degeneración Nerviosa/metabolismo , PPAR alfa/metabolismo , Animales , Regulación hacia Abajo , Fenofibrato/farmacología , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Hipolipemiantes/farmacología , Masculino , Degeneración Nerviosa/tratamiento farmacológico , PPAR alfa/genética , Ratas , Ratas Sprague-DawleyRESUMEN
Deficiency of endothelial progenitor cells, including endothelial colony-forming cells (ECFCs) and circulating angiogenic cells (CACs), plays an important role in retinal vascular degeneration in diabetic retinopathy (DR). Fenofibrate, an agonist of peroxisome proliferator-activated receptor α (PPARα), has shown therapeutic effects on DR in both patients and diabetic animal models. However, the function of PPARα in ECFC/CACs has not been defined. In this study, we determined the regulation of ECFC/CAC by PPARα. As shown by flow cytometry and Seahorse analysis, ECFC/CAC numbers and mitochondrial function were decreased in the bone marrow, circulation, and retina of db/db mice, correlating with PPARα downregulation. Activation of PPARα by fenofibrate normalized ECFC/CAC numbers and mitochondrial function in diabetes. In contrast, PPARα knockout exacerbated ECFC/CAC number decreases and mitochondrial dysfunction in diabetic mice. Primary ECFCs from PPARα -/- mice displayed impaired proliferation, migration, and tube formation. Furthermore, PPARα -/- ECFCs showed reduced mitochondrial oxidation and glycolysis compared with wild type, correlating with decreases of Akt phosphorylation and expression of its downstream genes regulating ECFC fate and metabolism. These findings suggest that PPARα is an endogenous regulator of ECFC/CAC metabolism and cell fate. Diabetes-induced downregulation of PPARα contributes to ECFC/CAC deficiency and retinal vascular degeneration in DR.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Retinopatía Diabética/metabolismo , Células Progenitoras Endoteliales/metabolismo , PPAR alfa/metabolismo , Retina/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Células Progenitoras Endoteliales/efectos de los fármacos , Fenofibrato/farmacología , Hipolipemiantes/farmacología , Masculino , Ratones , Ratones Noqueados , PPAR alfa/genética , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Retina/efectos de los fármacosRESUMEN
Fenofibrate is a peroxisome proliferator-activated receptor α (PPARα) agonist and has been shown to have therapeutic effects on diabetic retinopathy (DR). However, the effects of fenofibrate through systemic administration are not as potent as desired due to inefficient drug delivery to the retina. The present study aimed to explore the sustained therapeutic effects of fenofibrate-loaded biodegradable nanoparticles (NP) on both DR and neovascular age-related macular degeneration (AMD). Fenofibrate was successfully encapsulated into poly(lactic- co-glycolic acid) (PLGA) NP (Feno-NP), and Feno-NP were optimized by varying polymer composition to achieve high drug loading and prolonged drug release. The Feno-NP made of PLGA 34 kDa demonstrated a drug content of 6% w/w and a sustained drug release up to 60 days in vitro. Feno-NP (PLGA 34 kDa) was selected for following in vivo studies, and one single intravitreal (IVT) injection of Feno-NP into rat eyes with a 30G fine needle maintained sustained fenofibric acid drug level in the eye for more than 60 days. The efficacy of Feno-NP in DR and neovascular AMD was investigated using streptozotocin (STZ)-induced diabetic rats, laser-induced choroidal neovascularization (CNV) rats, and very low-density lipoprotein receptor knockout ( Vldlr -/-) mice. Therapeutic effects of Feno-NP were evaluated by measuring electroretinogram (ERG), retinal vascular leakage, leukostasis, CNV size, and retinal levels of vascular endothelial growth factor (VEGF) and intracellular adhesion molecule-1 (ICAM-1). In diabetic rats, Feno-NP ameliorated retinal dysfunctions, reduced retinal vascular leakage, inhibited retinal leukostasis, and downregulated the overexpression of VEGF and ICAM-1 at 8 weeks after one IVT injection. In addition, Feno-NP reduced retinal vascular leakage and CNV formation in both CNV rats and Vldlr -/- mice. Moreover, no toxicity of Feno-NP or Blank-NP to retinal structure and function was detected. Feno-NP exhibited good physiochemical characteristics and controlled drug release profile, conferring prolonged beneficial effects on DR and neovascular AMD.
Asunto(s)
Diabetes Mellitus Experimental/tratamiento farmacológico , Retinopatía Diabética/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/métodos , Fenofibrato/análogos & derivados , Hipolipemiantes/uso terapéutico , Nanopartículas/química , Degeneración Macular Húmeda/tratamiento farmacológico , Animales , Permeabilidad Capilar , Neovascularización Coroidal/tratamiento farmacológico , Diabetes Mellitus Experimental/inducido químicamente , Liberación de Fármacos , Fenofibrato/química , Fenofibrato/farmacocinética , Fenofibrato/uso terapéutico , Hipolipemiantes/química , Molécula 1 de Adhesión Intercelular/metabolismo , Leucostasis/tratamiento farmacológico , Ratones , Ratones Noqueados , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Ratas , Ratas Endogámicas BN , Retina/efectos de los fármacos , Retina/metabolismo , Estreptozocina/efectos adversos , Estreptozocina/farmacología , Distribución Tisular , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Diabetic retinopathy (DR) is a common neurovascular complication of type 1 diabetes. Current therapeutics target neovascularization characteristic of end-stage disease, but are associated with significant adverse effects. Targeting early events of DR such as neurodegeneration may lead to safer and more effective approaches to treatment. Two independent prospective clinical trials unexpectedly identified that the PPARα agonist fenofibrate had unprecedented therapeutic effects in DR, but gave little insight into the physiological and molecular mechanisms of action. The objective of the present study was to evaluate potential neuroprotective effects of PPARα in DR, and subsequently to identify the responsible mechanism of action. Here we reveal that activation of PPARα had a robust protective effect on retinal function as shown by Optokinetic tracking in a rat model of type 1 diabetes, and also decreased retinal cell death, as demonstrated by a DNA fragmentation ELISA. Further, PPARα ablation exacerbated diabetes-induced decline of visual function as demonstrated by ERG analysis. We further found that PPARα improved mitochondrial efficiency in DR, and decreased ROS production and cell death in cultured retinal neurons. Oxidative stress biomarkers were elevated in diabetic Pparα-/- mice, suggesting increased oxidative stress. Mitochondrially mediated apoptosis and oxidative stress secondary to mitochondrial dysfunction contribute to neurodegeneration in DR. Taken together, these findings identify a robust neuroprotective effect for PPARα in DR, which may be due to improved mitochondrial function and subsequent alleviation of energetic deficits, oxidative stress and mitochondrially mediated apoptosis.
Asunto(s)
Diabetes Mellitus Tipo 1/metabolismo , Retinopatía Diabética/metabolismo , Fármacos Neuroprotectores/metabolismo , PPAR alfa/metabolismo , Animales , Apoptosis/efectos de los fármacos , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Retinopatía Diabética/tratamiento farmacológico , Modelos Animales de Enfermedad , Fenofibrato/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estrés Oxidativo/efectos de los fármacos , Estudios Prospectivos , Ratas , Ratas Endogámicas BN , Ratas Sprague-Dawley , Retina/efectos de los fármacos , Retina/metabolismo , Enfermedades de la Retina/tratamiento farmacológico , Enfermedades de la Retina/metabolismoRESUMEN
PURPOSE: Our previous study demonstrated that Mab2F1, a murine monoclonal antibody blocking the Wnt/ß-catenin signaling pathway, has beneficial effects on experimental diabetic retinopathy and choroidal neovascularization (NV). The aforementioned antibody has been humanized. This study evaluated effects of the humanized antibody, H1L1, on NV. METHODS: H1L1 was evaluated in the alkali burn-induced corneal NV rat model. Rats with corneal NV were injected subconjunctivally with Mab2F1 or H1L1 using non-specific mouse or human IgG as controls. Corneal NV and opacity were evaluated using corneal NV area and inflammatory index. Expression of angiogenic and inflammatory factors and components of the Wnt/ß-catenin pathway in both the corneas of the animal model and human corneal epithelial (HCE) cells exposed to Wnt3a conditioned medium (WCM) were determined by Western blotting and a luciferase-based promoter assay. Cytotoxicities of these antibodies were evaluated by MTT assay. RESULTS: H1L1 reduced the area of corneal NV and opacity, similar to Mab2F1. Both Mab2F1 and H1L1 down-regulated the overexpression of angiogenic and inflammatory factors including VEGF, TNF-α and ICAM-1, and blocked the aberrant activation of the Wnt/ß-catenin pathway as shown by down-regulation of phosphorylated LRP6, total LRP6 and non-phosphorylated ß-catenin in the cornea of the NV model and cultured HCE cells exposed to WCM. Both antibodies also inhibited the transcriptional activity of ß-catenin induced by WCM in HCE cells. No toxic effects of the antibodies were observed in cultured HCE cells. CONCLUSIONS: H1L1 exhibits anti-angiogenic activities through blocking the Wnt/ß-catenin pathway.
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Inhibidores de la Angiogénesis/farmacología , Anticuerpos Monoclonales Humanizados/farmacología , Quemaduras Químicas/tratamiento farmacológico , Neovascularización de la Córnea/tratamiento farmacológico , Quemaduras Oculares/tratamiento farmacológico , Neovascularización Patológica , Vía de Señalización Wnt/efectos de los fármacos , Proteínas Angiogénicas/metabolismo , Animales , Quemaduras Químicas/metabolismo , Quemaduras Químicas/patología , Células Cultivadas , Neovascularización de la Córnea/inducido químicamente , Neovascularización de la Córnea/metabolismo , Neovascularización de la Córnea/patología , Modelos Animales de Enfermedad , Epitelio Corneal/efectos de los fármacos , Epitelio Corneal/metabolismo , Epitelio Corneal/patología , Quemaduras Oculares/inducido químicamente , Quemaduras Oculares/metabolismo , Quemaduras Oculares/patología , Humanos , Mediadores de Inflamación/metabolismo , Masculino , Ratas Sprague-Dawley , Hidróxido de SodioRESUMEN
BACKGROUND: Peroxisome proliferator activated receptor-alpha (PPARα) is a ubiquitously expressed nuclear receptor. The role of endogenous PPARα in retinal neuronal homeostasis is unknown. Retinal photoreceptors are the highest energy-consuming cells in the body, requiring abundant energy substrates. PPARα is a known regulator of lipid metabolism, and we hypothesized that it may regulate lipid use for oxidative phosphorylation in energetically demanding retinal neurons. RESULTS: We found that endogenous PPARα is essential for the maintenance and survival of retinal neurons, with Pparα -/- mice developing retinal degeneration first detected at 8 weeks of age. Using extracellular flux analysis, we identified that PPARα mediates retinal utilization of lipids as an energy substrate, and that ablation of PPARα ultimately results in retinal bioenergetic deficiency and neurodegeneration. This may be due to PPARα regulation of lipid transporters, which facilitate the internalization of fatty acids into cell membranes and mitochondria for oxidation and ATP production. CONCLUSION: We identify an endogenous role for PPARα in retinal neuronal survival and lipid metabolism, and furthermore underscore the importance of fatty acid oxidation in photoreceptor survival. We also suggest PPARα as a putative therapeutic target for age-related macular degeneration, which may be due in part to decreased mitochondrial efficiency and subsequent energetic deficits.
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Ácidos Grasos/metabolismo , Metabolismo de los Lípidos , PPAR alfa/genética , Retina/metabolismo , Neuronas Retinianas/fisiología , Animales , Ratones , Ratones Endogámicos C57BL , Oxidación-Reducción , PPAR alfa/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Purpose: Clinical studies have shown that peroxisome proliferator-activated receptor alpha (PPARα) agonist fenofibrate has therapeutic effects on diabetic retinopathy (DR). The purpose of this study was to identify a novel PPARα agonist and to evaluate its beneficial effects on DR. Methods: The transcriptional activity of PPARα was measured by a luciferase-based promoter assay. TUNEL was used to evaluate apoptosis in retinal precursor cells (R28). Diabetes was induced in rats by injection of streptozotocin. Retinal inflammation was examined using leukostasis assay, and retinal vascular leakage was measured using permeability assay. Retinal function was measured using electroretinogram (ERG) recording, and retinal apoptosis was quantified using the cell death ELISA. The anti-angiogenic effect was evaluated in the oxygen-induced retinopathy (OIR) model. Results: A compound, 7-chloro-8-methyl-2-phenylquinoline-4-carboxylic acid (Y-0452), with a chemical structure distinct from existing PPARα agonists, activated PPARα transcriptional activity and upregulated PPARα expression. Y-0452 significantly inhibited human retinal capillary endothelial cell migration and tube formation. The compound also protected R28 cells against apoptosis and inhibited NF-κB signaling in R28 cells exposed to palmitate. In diabetic rats, Y-0452 ameliorated leukostasis and vascular leakage in the retina. In addition, Y-0452 preserved the retinal function and reduced retinal cell death in diabetic rats. Y-0452 also alleviated retinal neovascularization in the OIR model. Conclusions: Y-0452 is a novel PPARα agonist and has therapeutic potential for DR.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Diabetes Mellitus Experimental/tratamiento farmacológico , Retinopatía Diabética/tratamiento farmacológico , Hidrocarburos Clorados/uso terapéutico , PPAR alfa/agonistas , Quinolinas/uso terapéutico , Neovascularización Retiniana/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Permeabilidad Capilar/efectos de los fármacos , Línea Celular , Movimiento Celular , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Retinopatía Diabética/genética , Retinopatía Diabética/patología , Modelos Animales de Enfermedad , Electrorretinografía , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica/fisiología , Etiquetado Corte-Fin in Situ , Leucostasis , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/antagonistas & inhibidores , Oxígeno/toxicidad , PPAR alfa/genética , PPAR alfa/metabolismo , Regiones Promotoras Genéticas , Ratas , Ratas Endogámicas BN , Neovascularización Retiniana/genética , Neovascularización Retiniana/patología , Vasos Retinianos/patología , Estreptozocina , Activación TranscripcionalRESUMEN
Purpose: This study was designed to evaluate effects of fenofibric acid (Feno-FA), a peroxisome proliferator-activated receptor-alpha (PPARα) agonist, on ocular neovascularization (NV) in models recapitulating neovascular age-related macular degeneration (AMD), and to explore whether the effects are PPARα dependent. Methods: Laser-induced choroidal NV (CNV) in rats and very low-density lipoprotein receptor knockout (Vldlr-/-) mice received daily intraperitoneal injections of Feno-FA or vehicle. Vascular leakage was examined by fundus fluorescein angiography and permeability assay using Evans blue as tracer. In CNV rats, severity of CNV was evaluated by CNV areas and CNV volume. In Vldlr-/- mice, subretinal NV (SRNV) and intraretinal NV (IRNV) were quantified in choroid flat mount and retina flat mount, respectively. Inflammatory factors were measured using Western blotting and retinal leukostasis assay. Further, Pparα-/- mice and age-matched wild-type (WT) mice were used for laser-induced CNV and treated with Feno-FA to explore the underlying mechanism. Results: Feno-FA significantly reduced vascular leakage in CNV rats and Vldlr-/- mice, reduced CNV volume in laser-induced CNV rats, and suppressed SRNV and IRNV in Vldlr-/- mice. In addition, Feno-FA downregulated the expression of inflammatory factors, including VEGF, TNF-α, and intercellular cell adhesion molecule-1 (ICAM-1), in the eyecups of CNV rats and decreased adherent retinal leukocytes in Vldlr-/- mice. Furthermore, Pparα-/- mice developed more severe CNV compared with WT mice, and PPARα knockout abolished the beneficial effects of Feno-FA on CNV. Conclusions: Feno-FA has therapeutic effects on ocular NV in models recapitulating neovascular AMD through a PPARα-dependent mechanism.
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Neovascularización Coroidal/tratamiento farmacológico , Modelos Animales de Enfermedad , Fenofibrato/análogos & derivados , Hipolipemiantes/uso terapéutico , PPAR alfa/agonistas , Degeneración Macular Húmeda/tratamiento farmacológico , Animales , Western Blotting , Permeabilidad Capilar , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Fenofibrato/uso terapéutico , Angiografía con Fluoresceína , Inyecciones Intraperitoneales , Molécula 1 de Adhesión Intercelular/metabolismo , Leucostasis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , PPAR alfa/genética , PPAR alfa/metabolismo , Ratas , Ratas Endogámicas BN , Receptores de LDL/genética , Receptores de LDL/metabolismo , Tomografía de Coherencia Óptica , Factor de Necrosis Tumoral alfa/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Degeneración Macular Húmeda/metabolismo , Degeneración Macular Húmeda/patologíaRESUMEN
Fenofibrate, a specific agonist of peroxisome proliferator-activated receptor-α (PPARα), displays robust therapeutic effects on diabetic retinopathy (DR) in patients with type 2 diabetes. Our recent studies have shown that PPARα is downregulated in the diabetic retina, which contributes to the pathogenesis of DR. However, the mechanism for diabetes-induced downregulation of PPARα remains unknown. We investigated the role of microRNA-21 (miR-21) in regulating PPARα in DR. miR-21 was overexpressed, while PPARα levels were decreased in the retina of db/db mice, a model of type 2 diabetes. Such alterations were also observed in palmitate-treated retinal endothelial cells. miR-21 targeted PPARα by inhibiting its mRNA translation. Knockout of miR-21 prevented the decrease of PPARα, alleviated microvascular damage, ameliorated inflammation, and reduced cell apoptosis in the retina of db/db mice. Intravitreal injection of miR-21 inhibitor attenuated PPARα downregulation and ameliorated retinal inflammation in db/db mice. Further, retinal miR-21 levels were increased, while PPARα levels were decreased in oxygen-induced retinopathy (OIR). Knockout of miR-21 prevented PPARα downregulation and ameliorated retinal neovascularization and inflammation in OIR retinas. In conclusion, diabetes-induced overexpression of miR-21 in the retina is at least partly responsible for PPARα downregulation in DR. Targeting miR-21 may represent a novel therapeutic strategy for DR.
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Diabetes Mellitus Tipo 2/genética , Retinopatía Diabética/genética , MicroARNs/genética , PPAR alfa/genética , Animales , Apoptosis/genética , Western Blotting , Línea Celular , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Retinopatía Diabética/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo , Humanos , Inflamación , Ratones , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la Polimerasa , Retina/metabolismo , Neovascularización Retiniana/genética , Epitelio Pigmentado de la Retina/citología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Members of the family of serine proteinase inhibitors, such as kallistatin, have been shown to inhibit canonical Wnt-TCF/LEF-ß-catenin signaling via their interactions with the Wnt co-receptor LRP6. Yet the effects of transgenic overexpression of anti-Wnt serpins on hematopoiesis and lymphopoiesis are not well known. We studied the effects of human kallistatin (SERPINA4) on Wnt reporter activity in various cell types throughout the hematopoietic system and associated impacts on circulating white blood cell profiles. Transgenic overexpression of kallistatin suppressed Wnt-TCF/LEF-ß-catenin signaling in bone marrow, as demonstrated using a Wnt reporter mouse. Further, kallistatin overexpression and treatment were associated with reduced Wnt-TCF/LEF-ß-catenin activity in CD34+ c-kit+ bone marrow cells and CD19+ B lymphocytes, with reduced levels of these populations in bone marrow and peripheral circulation, respectively. The presence of CD3+CD4+, CD3+CD8+, and CD3- NK1.1+ T lymphocytes were not significantly affected. Our data suggest that overexpression of kallistatin interferes with lymphopoiesis, ultimately impacting the level of circulating CD19+ B lymphocytes.
Asunto(s)
Antígenos CD19/inmunología , Linfocitos B/inmunología , Linfopoyesis/inmunología , Serpinas/inmunología , Proteínas Wnt/inmunología , Vía de Señalización Wnt/inmunología , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos CD/metabolismo , Antígenos CD19/genética , Antígenos CD19/metabolismo , Linfocitos B/citología , Linfocitos B/metabolismo , Pollos , Humanos , Ratones , Ratones Transgénicos , Serpinas/biosíntesis , Serpinas/genética , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Proteínas Wnt/antagonistas & inhibidores , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/genéticaRESUMEN
Current anti-VEGF drugs for patients with diabetic retinopathy suffer from short residence time in the vitreous of the eye. In order to maintain biologically effective doses of drug for inhibiting retinal neovascularization, patients are required to receive regular monthly injections of drug, which often results in low patient compliance and progression of the disease. To improve the intravitreal residence time of anti-VEGF drugs, we have synthesized multivalent bioconjugates of an anti-VEGF protein, soluble fms-like tyrosine kinase-1 (sFlt) that is covalently grafted to chains of hyaluronic acid (HyA), conjugates that are termed mvsFlt. Using a mouse corneal angiogenesis assay, we demonstrate that covalent conjugation to HyA chains does not decrease the bioactivity of sFlt and that mvsFlt is equivalent to sFlt at inhibiting corneal angiogenesis. In a rat vitreous model, we observed that mvsFlt had significantly increased intravitreal residence time compared to the unconjugated sFlt after 2 days. The calculated intravitreal half-lives for sFlt and mvsFlt were 3.3 and 35 hours, respectively. Furthermore, we show that mvsFlt is more effective than the unconjugated form at inhibiting retinal neovascularization in an oxygen-induced retinopathy model, an effect that is most likely due to the longer half-life of mvsFlt in the vitreous. Taken together, our results indicate that conjugation of sFlt to HyA does not affect its affinity for VEGF and this conjugation significantly improves drug half-life. These in vivo results suggest that our strategy of multivalent conjugation could substantially improve upon drug half-life, and thus the efficacy of currently available drugs that are used in diseases such as diabetic retinopathy, thereby improving patient quality of life.
Asunto(s)
Neovascularización de la Córnea/tratamiento farmacológico , Retinopatía Diabética/tratamiento farmacológico , Ácido Hialurónico/uso terapéutico , Neovascularización Retiniana/tratamiento farmacológico , Receptor 1 de Factores de Crecimiento Endotelial Vascular/uso terapéutico , Animales , Neovascularización de la Córnea/patología , Retinopatía Diabética/patología , Ácido Hialurónico/administración & dosificación , Masculino , Ratas , Neovascularización Retiniana/patología , Resultado del Tratamiento , Receptor 1 de Factores de Crecimiento Endotelial Vascular/administración & dosificaciónRESUMEN
Retinal Müller cells are major producers of inflammatory and angiogenic cytokines which contribute to diabetic retinopathy (DR). Over-activation of the Wnt/ß-catenin pathway has been shown to play an important pathogenic role in DR. However, the roles of Müller cell-derived Wnt/ß-catenin signaling in retinal neovascularization (NV) and DR remain undefined. In the present study, mice with conditional ß-catenin knockout (KO) in Müller cells were generated and subjected to oxygen-induced retinopathy (OIR) and streptozotocin (STZ)-induced diabetes. Wnt signaling was evaluated by measuring levels of ß-catenin and expression of its target genes using immunoblotting. Retinal vascular permeability was measured using Evans blue as a tracer. Retinal NV was visualized by angiography and quantified by counting pre-retinal nuclei. Retinal pericyte loss was evaluated using retinal trypsin digestion. Electroretinography was performed to examine visual function. No abnormalities were detected in the ß-catenin KO mice under normal conditions. In OIR, retinal levels of ß-catenin and VEGF were significantly lower in the ß-catenin KO mice than in littermate controls. The KO mice also had decreased retinal NV and vascular leakage in the OIR model. In the STZ-induced diabetic model, disruption of ß-catenin in Müller cells attenuated over-expression of inflammatory cytokines and ameliorated pericyte dropout in the retina. These findings suggest that Wnt signaling activation in Müller cells contributes to retinal NV, vascular leakage and inflammation and represents a potential therapeutic target for DR.