RESUMEN
Diabetes can result in impaired corneal wound healing. Mitochondrial dysfunction plays an important role in diabetic complications. However, the regulation of mitochondria function in the diabetic cornea and its impacts on wound healing remain elusive. The present study aimed to explore the molecular basis for the disturbed mitochondrial metabolism and subsequent wound healing impairment in the diabetic cornea. Seahorse analysis showed that mitochondrial oxidative phosphorylation is a major source of ATP production in human corneal epithelial cells. Live corneal biopsy punches from type 1 and type 2 diabetic mouse models showed impaired mitochondrial functions, correlating with impaired corneal wound healing, compared to nondiabetic controls. To approach the molecular basis for the impaired mitochondrial function, we found that Peroxisome Proliferator-Activated Receptor-α (PPARα) expression was downregulated in diabetic human corneas. Even without diabetes, global PPARα knockout mice and corneal epithelium-specific PPARα conditional knockout mice showed disturbed mitochondrial function and delayed wound healing in the cornea, similar to that in diabetic corneas. In contrast, fenofibrate, a PPARα agonist, ameliorated mitochondrial dysfunction and enhanced wound healing in the corneas of diabetic mice. Similarly, corneal epithelium-specific PPARα transgenic overexpression improved mitochondrial function and enhanced wound healing in the cornea. Furthermore, PPARα agonist ameliorated the mitochondrial dysfunction in primary human corneal epithelial cells exposed to diabetic stressors, which was impeded by siRNA knockdown of PPARα, suggesting a PPARα-dependent mechanism. These findings suggest that downregulation of PPARα plays an important role in the impaired mitochondrial function in the corneal epithelium and delayed corneal wound healing in diabetes.
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Diabetes Mellitus Experimental , PPAR alfa , Ratones , Humanos , Animales , PPAR alfa/genética , PPAR alfa/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Córnea/metabolismo , Cicatrización de Heridas/fisiología , Ratones Noqueados , Mitocondrias/metabolismoRESUMEN
Injuries to the retinal pigment epithelium (RPE) and outer retina often result in the accumulation of retinal microglia within the subretinal space. These subretinal microglia play crucial roles in inflammation and resolution, but the mechanisms governing their functions are still largely unknown. Our previous research highlighted the protective functions of choroidal γδ T cells in response to RPE injury. In the current study, we employed single-cell RNA sequencing approach to characterize the profiles of immune cells in mouse choroid. We found that γδ T cells were the primary producer of interleukin-17 (IL-17) in the choroid. IL-17 signaled through its receptor on the RPE, subsequently triggering the production of interleukin-6. This cascade of cytokines initiated a metabolic reprogramming of subretinal microglia, enhancing their capacity for lipid metabolism. RPE-specific knockout of IL-17 receptor A led to the dysfunction of subretinal microglia and RPE pathology. Collectively, our findings suggest that responding to RPE injury, the choroidal γδ T cells can initiate a protective signaling cascade that ensures the proper functioning of subretinal microglia.
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Degeneración Macular , Degeneración Retiniana , Animales , Ratones , Citocinas/metabolismo , Interleucina-17/genética , Interleucina-17/metabolismo , Degeneración Macular/patología , Retina/metabolismo , Degeneración Retiniana/metabolismo , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
The mechanistic target of rapamycin (mTOR) is assembled into signaling complexes of mTORC1 or mTORC2, and plays key roles in cell metabolism, stress response, and nutrient and growth factor sensing. Accumulating evidence from human and animal model studies has demonstrated a pathogenic role of hyperactive mTORC1 in age-related macular degeneration (AMD). The retinal pigment epithelium (RPE) is a primary injury site in AMD. In mouse models of RPE-specific deletion of Tuberous sclerosis 1 (Tsc1), which encodes an upstream suppressor of mTORC1, the hyperactivated mTORC1 metabolically reprogrammed the RPE and led to the degeneration of the outer retina and choroid (CH). In the current study, we use single-cell RNA sequencing (scRNA-seq) to identify an RPE mTORC1 downstream protein, dopamine- and cyclic AMP-regulated phosphoprotein of molecular weight 32,000 (DARPP-32). DARPP-32 was not found in healthy RPE but localized to drusen and basal linear deposits in human AMD eyes. In animal models, overexpressing DARPP-32 by adeno-associated virus (AAV) led to abnormal RPE structure and function. The data indicate that DARPP-32 is a previously unidentified signaling protein subjected to mTORC1 regulation and may contribute to RPE degeneration in AMD.
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Fosfoproteína 32 Regulada por Dopamina y AMPc , Degeneración Macular , Diana Mecanicista del Complejo 1 de la Rapamicina , Epitelio Pigmentado de la Retina , Animales , Modelos Animales de Enfermedad , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Activación Enzimática , Humanos , Degeneración Macular/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Transducción de SeñalRESUMEN
In ischemic retinopathy, overactivated retinal myeloid cells are a crucial driving force of pathological angiogenesis and inflammation. The cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) signaling are key regulators of inflammation. This study aims to investigate the association of cGAS-STING signaling with ischemic retinopathy and the regulation of its activation. We found that protein levels of cGAS and STING were markedly up-regulated in retinal myeloid cells isolated from mice with oxygen-induced retinopathy (OIR). Knockout of Sting and pharmacological inhibition of STING both alleviated retinal neovascularization (NV) and reduced retinal vascular leakage in OIR. Further, Sting knockout and STING inhibitor also alleviated leukocyte adhesion to retinal vasculature and infiltration into the retina as well as microglial activation in OIR. These results suggest that cGAS-STING signaling played a pathogenic role in retinal myeloid cell activation and NV in ischemic retinopathy. To identify the regulation of cGAS-STING signaling in OIR, we evaluated the role of transcription factor peroxisome proliferator-activated receptor α (PPARα). The results demonstrated that PPARα was down-regulated in OIR retinas, primarily in myeloid cells. Furthermore, Pparα knockout significantly up-regulated cGAS and STING levels in retinal CD11b+ cells, while PPARα agonist inhibited cGAS-STING signaling and cytosolic mitochondrial DNA (mtDNA) release, a causative feature for cGAS activation. Knockout of Sting ameliorated retinal NV, hyperpermeability, and leukostasis in Pparα-/- mice with OIR. These observations suggest that PPARα regulates cGAS-STING signaling, likely through mtDNA release, and thus, is a potential therapeutic target for ischemic retinopathy.
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PPAR alfa , Enfermedades de la Retina , Animales , Ratones , Modelos Animales de Enfermedad , ADN Mitocondrial , Inflamación , Isquemia/complicaciones , Proteínas de la Membrana/metabolismo , Ratones Noqueados , Neovascularización Patológica , Nucleotidiltransferasas/metabolismo , PPAR alfa/genética , Enfermedades de la Retina/genéticaRESUMEN
SignificanceIn humans, genetic mutations in the retinal pigment epithelium (RPE) 65 are associated with blinding diseases, for which there is no effective therapy alleviating progressive retinal degeneration in affected patients. Our findings uncovered that the increased free opsin caused by enhancing the ambient light intensity increased retinal activation, and when compounded with the RPE visual cycle dysfunction caused by the heterozygous D477G mutation and aggregation, led to the onset of retinal degeneration.
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Proteínas del Ojo , Genes Dominantes , Distrofias Retinianas , cis-trans-Isomerasas , Animales , Proteínas del Ojo/genética , Ratones , Ratones Noqueados , Mutación , Retina/enzimología , Retina/patología , Distrofias Retinianas/genética , Visión Ocular , cis-trans-Isomerasas/genéticaRESUMEN
Corneal dysfunctions associated with Diabetes Mellitus (DM), termed diabetic keratopathy (DK), can cause impaired vision and/or blindness. Hypoxia affects both Type 1 (T1DM) and Type 2 (T2DM) surprisingly, the role of hypoxia in DK is unexplored. The aim of this study was to examine the impact of hypoxia in vitro on primary human corneal stromal cells derived from Healthy (HCFs), and diabetic (T1DMs and T2DMs) subjects, by exposing them to normoxic (21% O2) or hypoxic (2% O2) conditions through 2D and 3D in vitro models. Our data revealed that hypoxia affected T2DMs by slowing their wound healing capacity, leading to significant alterations in oxidative stress-related markers, mitochondrial health, cellular homeostasis, and endoplasmic reticulum health (ER) along with fibrotic development. In T1DMs, hypoxia significantly modulated markers related to membrane permeabilization, oxidative stress via apoptotic marker (BAX), and protein degradation. Hypoxic environment induced oxidative stress (NOQ1 mediated reduction of superoxide in T1DMs and Nrf2 mediated oxidative stress in T2DMs), modulation in mitochondrial health (Heat shock protein 27 (HSP27), and dysregulation of cellular homeostasis (HSP90) in both T1DMs and T2DMs. This data underscores the significant impact of hypoxia on the diabetic cornea. Further studies are warranted to delineate the complex interactions.
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Enfermedades de la Córnea , Diabetes Mellitus , Humanos , Sustancia Propia/metabolismo , Córnea/metabolismo , Enfermedades de la Córnea/etiología , Enfermedades de la Córnea/metabolismo , Hipoxia/metabolismoRESUMEN
BACKGROUND: Previous reports have indicated that disrupting the Wnt/ß-catenin pathway in dendritic cells (DCs) may affect the progression of autoimmune inflammation; however, the factors and timing that regulate Wnt/ß-catenin signaling have not been clearly understood. METHODS: Experimental autoimmune uveitis (EAU) mice and Vogt-Koyanagi-Harada disease (VKH) patient samples were used to detect the expression of Wnt/ß-catenin pathway genes. Western blot, real-time PCR, flow cytometry, and ELISA were performed to examine the expression of components of the Wnt/ß-catenin pathway and inflammatory factors. DC-specific ß-catenin knockout mice and 6-bromoindirubin-3'-oxime (BIO) administered mice were used to observe the effect of disrupting the Wnt pathway on EAU pathogenesis. RESULTS: Wnt/ß-catenin signaling was inhibited in DCs during the induction phase of EAU. The inhibition was mediated by pertussis toxin (PTX), which promoted DC maturation, in turn promoting pathogenic T cell proliferation and differentiation. In vivo experiments confirmed that deleting ß-catenin in DCs enhanced EAU severity, and pre-injection of PTX advanced EAU onset. Administration of a Wnt activator (BIO) limited the effects of PTX, in turn ameliorating EAU. CONCLUSIONS: Our results demonstrate that PTX plays a key role as a virulence factor in initiating autoimmune inflammation via DCs by inhibiting Wnt/ß-catenin signaling in EAU, and highlight the potential mechanism by which infection can trigger apparent autoimmunity.
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Enfermedades Autoinmunes , Uveítis , Ratones , Animales , Toxina del Pertussis/toxicidad , Autoinmunidad , Vía de Señalización Wnt , beta Catenina/metabolismo , Uveítis/inducido químicamente , Uveítis/tratamiento farmacológico , Inflamación/metabolismo , Células DendríticasRESUMEN
Prolonged hyperglycemia during diabetes mellitus (DM) is associated with severe complications that may affect both the anterior and posterior ocular segments, leading to impaired vision or blindness. The cornea is a vital part of the eye that has a dual role as a protective transparent barrier and as a major refractive structure and is likewise negatively affected by hyperglycemia in DM. Understanding the cellular and molecular mechanisms underlying the phenotypic changes associated with DM is critical to developing targeted therapies to promote tissue integrity. In this proof-of-concept study, we applied a cell sheet-based approach to generate stacked constructs of physiological corneal thickness using primary human corneal fibroblasts isolated from cadaveric control (healthy), Type 1 DM and Type 2 DM corneal tissues. Self-assembled corneal stromal sheets were generated after 2 weeks in culture, isolated, and subsequently assembled to create stacked constructs, which were evaluated using transmission electron microscopy. Analysis of gene expression patterns revealed significant downregulation of fibrotic markers, α-smooth muscle actin, and collagen type 3, with stacking in Type 2 DM constructs when compared to controls. IGF1 expression was significantly upregulated in Type 2 DM constructs compared to controls with a significant reduction induced by stacking. This study describes the development of a thicker, self-assembled corneal stromal construct as a platform to evaluate phenotypic differences associated with DM-derived corneal fibroblasts and enable the development of targeted therapeutics to promote corneal integrity.
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Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Hiperglucemia , Humanos , Sustancia Propia/metabolismo , Córnea , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Hiperglucemia/metabolismoRESUMEN
The retina pigmented epithelium 65 kDa protein (RPE65) is an essential enzyme in the visual cycle that regenerates the 11-cis-retinal chromophore obligatory for vision. Mutations in RPE65 are associated with blinding diseases. D477G (C.1430G > A) is the only known RPE65 variant to cause autosomal dominant retinitis pigmentosa (adRP). Previously, we reported that the heterozygous D477G knock-in (WT/KI) mice exposed to dim light intensity demonstrated delayed chromophore regeneration rates and slowed recovery of photoreceptor sensitivity following photobleaching. However, visual function and retinal architecture were indistinguishable from the wild-type (WT) mice. In this study, when maintained under the physiological day-light intensity (2 K lux), the WT/KI heterozygous mice displayed retina degeneration and reduced electroretinography (ERG) amplitude, recapitulating that observed in human patients. Our findings indicated the importance of the light environment in the mechanism of RPE65 D477G pathogenicity.
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Degeneración Retiniana , cis-trans-Isomerasas , Humanos , Ratones , Animales , Modelos Animales de Enfermedad , cis-trans-Isomerasas/genética , Retina/metabolismo , Mutación , Electrorretinografía , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , EpitelioRESUMEN
Very low-density lipoprotein receptor (VLDLR) is a multifunctional transmembrane protein. Beyond the function of the full-length VLDLR in lipid transport, the soluble ectodomain of VLDLR (sVLDLR) confers anti-inflammatory and antiangiogenic roles in ocular tissues through inhibition of canonical Wnt signaling. However, it remains unknown how sVLDLR is shed into the extracellular space. In this study, we present the first evidence that a disintegrin and metalloprotease 17 (ADAM17) is responsible for sVLDLR shedding in human retinal pigment epithelium cells using pharmacological and genetic approaches. Among selected proteinase inhibitors, an ADAM17 inhibitor demonstrated the most potent inhibitory effect on sVLDLR shedding. siRNA-mediated knockdown or CRISPR/Cas9-mediated KO of ADAM17 diminished, whereas plasmid-mediated overexpression of ADAM17 promoted sVLDLR shedding. The amount of shed sVLDLR correlated with an inhibitory effect on the Wnt signaling pathway. Consistent with these in vitro findings, intravitreal injection of an ADAM17 inhibitor reduced sVLDLR levels in the extracellular matrix in the mouse retina. In addition, our results demonstrated that ADAM17 cleaved VLDLR only in cells coexpressing these proteins, suggesting that shedding occurs in a cis manner. Moreover, our study demonstrated that aberrant activation of Wnt signaling was associated with decreased sVLDLR levels, along with downregulation of ADAM17 in ocular tissues of an age-related macular degeneration model. Taken together, our observations reveal the mechanism underlying VLDLR cleavage and identify a potential therapeutic target for the treatment of disorders associated with dysregulation of Wnt signaling.
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Proteína ADAM17/metabolismo , Degeneración Macular/metabolismo , Receptores de LDL/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Vía de Señalización Wnt , Proteína ADAM17/antagonistas & inhibidores , Proteína ADAM17/genética , Animales , Modelos Animales de Enfermedad , Humanos , Degeneración Macular/genética , Ratones , Ratones Noqueados , Dominios Proteicos , Receptores de LDL/genéticaRESUMEN
The purpose of the study was to investigate the role of Prolactin-Induced Protein (PIP) in corneal wound healing, in vivo and in vitro. In C57BL/6J mice, corneal epithelia was removed using an ocular burr. Phosphate buffered saline (PBS) or PIP (0.5 and 1.0 µg/mL) was applied topically or subconjunctivally injected. PIP accelerated wound closure as early as 24 h. PIP treatment promoted corneal wound healing and epithelial integrity and thickness. Integrin α6, integrin ß4, Thrombospondin-1, and TGF-ß1 expressions were all downregulated by PIP after wound closure. In vitro, scratch assays were performed using primary human epithelial cells (HCECs) and human corneal fibroblasts (HCFs), stimulated with PIP at various dosages. PIP treatment promoted both HCECs and HCFs migration. PIP upregulated expression of integrin α6, integrin ß4, and Thrombospondin-1 in HCECs. Expression of TGF-ß1 in HCECs and expression of smooth muscle actin (SMA) and Type III Collagen (Col III) in HCFs were significantly downregulated at 150 ng/mL PIP. PIP exhibits noteworthy anti-fibrotic potentiality. While the mechanism of how PIP is impactful on the corneal wound healing cascade is unknown, our findings are novel and further studies are warranted in order to unravel any therapeutic potential.
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Lesiones de la Cornea , Factor de Crecimiento Transformador beta1 , Ratones , Animales , Humanos , Factor de Crecimiento Transformador beta1/farmacología , Factor de Crecimiento Transformador beta1/metabolismo , Prolactina/farmacología , Integrina alfa6 , Ratones Endogámicos C57BL , Cicatrización de Heridas/fisiología , TrombospondinasRESUMEN
Subretinal fibrosis is a key pathological feature in neovascular age-related macular degeneration (nAMD). Previously, we identified soluble very low-density lipoprotein receptor (sVLDLR) as an endogenous Wnt signaling inhibitor. This study investigates whether sVLDLR plays an anti-fibrogenic role in nAMD models, including Vldlr-/- mice and laser-induced choroidal neovascularization (CNV). We found that fibrosis factors including P-Smad2/3, α-SMA, and CTGF were upregulated in the subretinal area of Vldlr-/- mice and the laser-induced CNV model. The antibody blocking Wnt co-receptor LRP6 significantly attenuated the overexpression of fibrotic factors in these two models. Moreover, there was a significant reduction of sVLDLR in the interphotoreceptor matrix (IPM) in the laser-induced CNV model. A transgenic strain (sVLDLR-Tg) with sVLDLR overexpression in the IPM was generated. Overexpression of sVLDLR ameliorated the profibrotic changes in the subretinal area of the laser-induced CNV model. In addition, Wnt and TGF-ß signaling synergistically promoted fibrogenesis in human primary retinal pigment epithelium (RPE) cells. CRISPR/Cas9-mediated LRP6 gene knockout (KO) attenuated this synergistic effect. The disruption of VLDLR expression promoted, while the overexpression of sVLDLR inhibited TGF-ß-induced fibrosis. These findings suggest that overactivated Wnt signaling enhances the TGF-ß pathway in subretinal fibrosis. sVLDLR confers an antifibrotic effect, at least partially, through the inhibition of Wnt signaling and thus, has therapeutic potential for fibrosis.
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Neovascularización Coroidal/complicaciones , Modelos Animales de Enfermedad , Fibrosis/prevención & control , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Degeneración Macular/complicaciones , Receptores de LDL/fisiología , Epitelio Pigmentado de la Retina/patología , Animales , Sistemas CRISPR-Cas , Células Cultivadas , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Fibrosis/etiología , Fibrosis/metabolismo , Fibrosis/patología , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/antagonistas & inhibidores , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Epitelio Pigmentado de la Retina/metabolismo , Proteínas Smad/genética , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Vía de Señalización WntRESUMEN
In this contribution, we review models for the lattice effects in quantum dynamics calculations on surface scattering, which is important to modeling heterogeneous catalysis for achieving an interpretation of experimental measurements. Unlike dynamics models for reactions in the gas phase, those for heterogeneous reactions have to include the effects of the surface. For manageable computational costs in calculations, the effects of static surface (SS) are firstly modeled as this is simply and easily implemented. Then, the SS model has to be improved to include the effects of the flexible surface, that is the lattice effects. To do this, various surface models have been designed where the coordinates of the surface atoms are introduced in the Hamiltonian operator, especially those of the top surface atom. Based on this model Hamiltonian operator, extensive multi-dimension quantum dynamics calculations can be performed to recover the lattice effects. Here, we first review an overview of the techniques in constructing the Hamiltonian operator, which is a sum of the kinetic energy operator (KEO) and potential energy surface (PES). Since the PES containing the coordinates of the surface atoms in a cell is still expensive, the SS model is often accepted. We consider a mathematical model, called the coupled harmonic oscillator (CHO) model, to introduce the concepts of adiabatic and diabatic representations for separating the molecule and surface. Under the adiabatic model, we further introduce the expansion model where the potential function is Taylor expanded around the optimized geometry of the surface. By an expansion model truncated at the first and second order, various coupling surface models between the molecule and surface are derived. Moreover, by further and deeply understanding the adiabatic representation, an effective Hamiltonian operator is obtained by optimizing the total wave function in factorized form. By this factorized form of wave function and effective Hamiltonian operator, the geometry phase of the surface wave function is theoretically found. This theoretical prediction may be measured by carefully designing experiments. Finally, discussions on the adiabatic representation, the PES construction, and possibility of the classical-dynamics solutions are given. Based on these discussions, a simple outlook on the dynamics of photocatalytics is finally given.
RESUMEN
Dysregulation of Wnt signaling is implicated in multiple ocular disorders. The roles of Wnt co-receptors LRP5 and LRP6 in Wnt signaling regulation remain elusive, as most retinal cells express both of the co-receptors. To address this question, LRP5 and LRP6 were individually knocked-out in a human retinal pigment epithelium cell line using the CRISPR-Cas9 technology. Wnt signaling activity induced by various Wnt ligands was measured using wild-type and the KO cell lines. The results identified three groups of Wnt ligands based on their co-receptor specificity: 1) activation of Wnt signaling only through LRP6, 2) through both LRP5 and LRP6 and 3) predominantly through LRP5. These results indicate that LRP5 and LRP6 have differential roles in Wnt signaling regulation.
Asunto(s)
Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Vía de Señalización Wnt , Animales , Sistemas CRISPR-Cas , Línea Celular , Medios de Cultivo Condicionados , Receptores Frizzled/metabolismo , Técnicas de Inactivación de Genes , Humanos , Ligandos , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/deficiencia , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/deficiencia , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Ratones , Epitelio Pigmentado de la Retina/citología , Retinoides/metabolismo , Retinoides/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Proteína Wnt3A/metabolismoRESUMEN
Diabetic retinopathy (DR) is a common complication of diabetes and a leading cause of blindness among the working-age population. Diabetic patients often experience functional deficits in dark adaptation, contrast sensitivity, and color perception before any microvascular pathologies on the fundus become detectable. Previous studies showed that the regeneration of 11-cis-retinal and visual pigment is impaired in a type 1 diabetes animal model, which negatively affects visual function at the early stage of DR. Here, Akita mice, type 1 diabetic model, were treated with the visual pigment chromophore, 9-cis-retinal. This treatment rescued a- and b-wave amplitudes of scotopic electroretinography responses, compared with vehicle-treated Akita mice. In addition, the administration of 9-cis-retinal alleviated oxidative stress significantly as shown by reduced 3-nitrotyrosine levels in the retina of Akita mice. Furthermore, the 9-cis-retinal treatment decreased retinal apoptosis as shown by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and DNA fragment enzyme-linked immunosorbent assay. Overall, these findings showed that 9-cis-retinal administration restored visual pigment formation and decreased oxidative stress and retinal degeneration, which resulted in improved visual function in diabetic mice, suggesting that chromophore deficiency plays a causative role in visual defects in early DR.
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Retinopatía Diabética/fisiopatología , Diterpenos/farmacología , Retina/efectos de los fármacos , Retinaldehído/farmacología , Animales , Apoptosis/efectos de los fármacos , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Tipo 1/complicaciones , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Estrés Oxidativo/efectos de los fármacos , Retina/fisiopatologíaRESUMEN
Peroxisome Proliferator-Activated Receptors (PPARs) are a family of nuclear receptors that play essential roles in modulating cell differentiation, inflammation, and metabolism. Three subtypes of PPARs are known: PPAR-alpha (PPARα), PPAR-gamma (PPARγ), and PPAR-beta/delta (PPARß/δ). PPARα activation reduces lipid levels and regulates energy homeostasis, activation of PPARγ results in regulation of adipogenesis, and PPARß/δ activation increases fatty acid metabolism and lipolysis. PPARs are linked to various diseases, including but not limited to diabetes, non-alcoholic fatty liver disease, glaucoma and atherosclerosis. In the past decade, numerous studies have assessed the functional properties of PPARs in the eye and key PPAR mechanisms have been discovered, particularly regarding the retina and cornea. PPARγ and PPARα are well established in their functions in ocular homeostasis regarding neuroprotection, neovascularization, and inflammation, whereas PPARß/δ isoform function remains understudied. Naturally, studies on PPAR agonists and antagonists, associated with ocular pathology, have also gained traction with the development of PPAR synthetic ligands. Studies on PPARs has significantly influenced novel therapeutics for diabetic eye disease, ocular neuropathy, dry eye, and age-related macular degeneration (AMD). In this review, therapeutic potentials and implications will be highlighted, as well as reported adverse effects. Further investigations are necessary before any of the PPARs ligands can be utilized, in the clinics, to treat eye diseases. Future research on the prominent role of PPARs will help unravel the complex mechanisms involved in order to prevent and treat ocular diseases.
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Oftalmopatías/metabolismo , Metabolismo de los Lípidos/fisiología , Receptores Activados del Proliferador del Peroxisoma/fisiología , Animales , Homeostasis , Humanos , LigandosRESUMEN
Kallistatin is an inhibitor of tissue kallikrein and also inhibits the Wnt pathway. Its role in diabetic nephropathy (DN) is uncertain. Here we reported that serum kallistatin levels were significantly increased in diabetic patients with DN compared to those in diabetic patients without DN and healthy controls, and positively correlated with urinary albumin excretion. In addition, renal kallistatin levels were significantly upregulated in mouse models of type 1 (Akita, OVE26) and type 2 diabetes (db/db). To unveil the effects of kallistatin on DN and its underlying mechanism, we crossed transgenic mice overexpressing kallistatin with OVE26 mice (KS-tg/OVE). Kallistatin overexpression exacerbated albuminuria, renal fibrosis, inflammation, and oxidative stress in diabetes. Kallikrein activity was inhibited while the renin-angiotensin system (RAS) upregulated in the kidney of KS-tg/OVE mice compared to WT/OVE mice, suggesting a disturbed balance between the RAS and kallikrein-kinin systems. As shown by immunostaining of endothelial makers, renal vascular densities were decreased accompanied by increased HIF-1α and erythropoietin levels in the kidneys of KS-tg/OVE mice. Taken together, high levels of kallistatin exacerbate DN at least partly by inducing RAS overactivation and hypoxia. The present study demonstrated a positive correlation between kallistatin levels and DN, suggesting a potential biomarker for prognosis of DN.
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Diabetes Mellitus Tipo 2/metabolismo , Nefropatías Diabéticas/metabolismo , Sistema Renina-Angiotensina/fisiología , Serpinas/metabolismo , Regulación hacia Arriba/fisiología , Animales , Biomarcadores/metabolismo , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Modelos Animales de Enfermedad , Fibrosis/metabolismo , Humanos , Inflamación/metabolismo , Riñón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Estrés Oxidativo/fisiologíaRESUMEN
AIMS/HYPOTHESIS: Diabetes mellitus erectile dysfunction (DMED) is a common complication of diabetes. The level of pigment epithelium-derived factor (PEDF) is significantly upregulated in the serum of individuals with obesity and diabetes. However, whether elevated PEDF levels contribute to DMED remains unknown. This study aimed to investigate the pathogenic role of PEDF and its related mechanism in DMED. METHODS: We enrolled 65 men, of whom 20 were nondiabetic control participants, 21 participants with diabetes but without erectile dysfunction, and 24 with DMED. The International Index of Erectile Function (IIEF-5) questionnaire was administered to evaluate erectile function. Plasma PEDF in diabetic participants and streptozotocin (STZ)-induced diabetic animals was detected by ELISA. Erectile function was evaluated by measuring the intracavernous pressure (ICP) and the ICP/mean arterial pressure (MAP) ratio in STZ-induced diabetic rats treated with PEDF-neutralising antibody (PEDF-Ab), db/db mice treated with PEDF-Ab, and Pedf knockout mice with STZ-induced diabetes. The overexpression of PEDF was implemented by intraperitoneal injection of recombinant PEDF and intracavernous injection of PEDF-expressing adenovirus. A mechanistic study was performed by immunofluorescence staining, bimolecular fluorescence complementation (BiFC), immunoprecipitation and western blotting. RESULTS: We found that the plasma level of PEDF was significantly higher in participants with DMED compared with diabetic counterparts without erectile dysfunction and nondiabetic controls. Interestingly, PEDF levels were negatively correlated with plasma nitrite/nitrate levels and erectile function in DMED patients and STZ-induced diabetic rats. Furthermore, overexpression of PEDF significantly suppressed ICP and endothelial nitric oxide synthase (eNOS) phosphorylation in control rats. In contrast, the PEDF-Ab and Pedf knockout ameliorated ICP and eNOS phosphorylation in diabetic rats and mice. Mechanistically, PEDF promoted the membrane translocation of Hsp90ß and directly bound to the amino acid residues 341-724 of Hsp90ß on the endothelial cell surface, subsequently blocking intracellular Hsp90ß/Akt/eNOS complex formation and downregulating eNOS phosphorylation. CONCLUSIONS/INTERPRETATION: These results indicate that elevated PEDF levels contribute to impaired erectile function by suppressing Hsp90ß-mediated eNOS phosphorylation and that PEDF may represent a novel therapeutic target for diabetic erectile dysfunction. Graphical abstract.
Asunto(s)
Complicaciones de la Diabetes/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus/metabolismo , Disfunción Eréctil/metabolismo , Proteínas del Ojo/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Serpinas/metabolismo , Adulto , Animales , Anticuerpos Neutralizantes/farmacología , Estudios de Casos y Controles , Complicaciones de la Diabetes/genética , Diabetes Mellitus Experimental/complicaciones , Disfunción Eréctil/etiología , Disfunción Eréctil/genética , Proteínas del Ojo/genética , Técnicas de Sustitución del Gen , Humanos , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Factores de Crecimiento Nervioso/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Ratas , Serpinas/genéticaRESUMEN
Endothelial progenitor cells (EPCs) contribute to blood vessel formation. Canonical Wnt signaling plays an important role in physiological and pathological angiogenesis and EPC fate regulation. However, the mechanism for Wnt signaling to regulate EPC fate in neovascularization (NV) has not been clearly defined. Here, we showed that very low-density lipoprotein receptor knockout (Vldlr -/- ) mice, a model of ocular NV induced by Wnt signaling overactivation, have increased EPC numbers in the bone marrow, blood, and retina, as well as an elevated mitochondrial membrane potential indicating higher mitochondrial function of EPCs in the circulation. Isolated EPCs from Vldlr -/- mice showed overactivated Wnt signaling, correlating with increased mitochondrial function, mass, and DNA copy numbers, compared with WT EPCs. Our results also demonstrated that Wnt signaling upregulated mitochondrial biogenesis and function, while inhibiting glycolysis in EPCs, which further decreased EPC stemness and promoted EPCs to a more active state toward differentiation, which may contribute to pathologic vascular formation. Fenofibric acid, an active metabolite of fenofibrate, inhibited Wnt signaling and mitochondrial function in EPCs and decreased EPC numbers in Vldlr -/- mice. It also decreased mitochondrial biogenesis and reactive oxygen species production in Vldlr -/- EPCs, which may be responsible for its therapeutic effect on diabetic retinopathy. These findings demonstrated that Wnt signaling regulates EPC fate through metabolism, suggesting potential application of the EPC metabolic profile as predictor and therapeutic target for neovascular diseases. Stem Cells 2019;37:1331-1343.
Asunto(s)
Células Madre/metabolismo , Vía de Señalización Wnt/fisiología , Animales , Diferenciación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Células Progenitoras Endoteliales/metabolismo , Metaboloma , Ratones , Neovascularización Patológica/metabolismoRESUMEN
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.