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How reparative processes are coordinated following injury is incompletely understood. In recent studies, we showed that autocrine C3a and C5a receptor (C3ar1 and C5ar1) G protein-coupled receptor signaling plays an obligate role in vascular endothelial growth factor receptor 2 growth signaling in vascular endothelial cells. We documented the same interconnection for platelet-derived growth factor receptor growth signaling in smooth muscle cells, epidermal growth factor receptor growth signaling in epidermal cells, and fibroblast growth factor receptor signaling in fibroblasts, indicative of a generalized cell growth regulatory mechanism. In this study, we examined one physiological consequence of this signaling circuit. We found that disabling CD55 (also known as decay accelerating factor), which lifts restraint on autocrine C3ar1/C5ar1 signaling, concomitantly augments the growth of each cell type. The mechanism is heightened C3ar1/C5ar1 signaling resulting from the loss of CD55's restraint jointly potentiating growth factor production by each cell type. Examination of the effect of lifted CD55 restraint in four types of injury (burn, corneal denudation, ear lobe puncture, and reengraftment of autologous skin) showed that disabled CD55 function robustly accelerated healing in all cases, whereas disabled C3ar1/C5ar1 signaling universally retarded it. In wild-type mice with burns or injured corneas, applying a mouse anti-mouse CD55 blocking Ab (against CD55's active site) to wounds accelerated the healing rate by 40-70%. To our knowledge, these results provide new insights into mechanisms that underlie wound repair and open up a new tool for accelerating healing.
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Antígenos CD55 , Células Endoteliais , Fator A de Crescimento do Endotélio Vascular , Cicatrização , Animais , Camundongos , Células Endoteliais/metabolismo , Transdução de Sinais , Pele , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/fisiologia , Antígenos CD55/antagonistas & inibidores , Antígenos CD55/metabolismoRESUMO
Vascular inflammation is known to cause degeneration of retinal capillaries in early diabetic retinopathy (DR), a major microvascular complication of diabetes. Past studies investigating these diabetes-induced retinal vascular abnormalities have focused primarily on the role of molecular or biochemical cues. Here we show that retinal vascular inflammation and degeneration in diabetes are also mechanically regulated by the increase in retinal vascular stiffness caused by overexpression of the collagen-cross-linking enzyme lysyl oxidase (LOX). Treatment of diabetic mice with LOX inhibitor ß-aminopropionitrile (BAPN) prevented the increase in retinal capillary stiffness, vascular intracellular adhesion molecule-1 overexpression, and leukostasis. Consistent with these anti-inflammatory effects, BAPN treatment of diabetic mice blocked the upregulation of proapoptotic caspase-3 in retinal vessels, which concomitantly reduced retinal capillary degeneration, pericyte ghost formation, and the diabetes-induced loss of contrast sensitivity in these mice. Finally, our in vitro studies indicate that retinal capillary stiffening is sufficient to increase the adhesiveness and neutrophil elastase-induced death of retinal endothelial cells. By uncovering a link between LOX-dependent capillary stiffening and the development of retinal vascular and functional defects in diabetes, these findings offer a new insight into DR pathogenesis that has important translational potential.
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Diabetes Mellitus Experimental , Retinopatia Diabética , Degeneração Retiniana , Camundongos , Animais , Células Endoteliais , Diabetes Mellitus Experimental/complicações , Aminopropionitrilo/farmacologia , Retina/patologia , Retinopatia Diabética/patologia , Inflamação/patologia , Vasos Retinianos/patologia , Camundongos Endogâmicos C57BLRESUMO
Chronic, progressive retinal diseases, such as age-related macular degeneration (AMD), diabetic retinopathy, and retinitis pigmentosa, arise from genetic and environmental perturbations of cellular and tissue homeostasis. These disruptions accumulate with repeated exposures to stress over time, leading to progressive visual impairment and, in many cases, legal blindness. Despite decades of research, therapeutic options for the millions of patients suffering from these disorders remain severely limited, especially for treating earlier stages of pathogenesis when the opportunity to preserve the retinal structure and visual function is greatest. To address this urgent, unmet medical need, we employed a systems pharmacology platform for therapeutic development. Through integrative single-cell transcriptomics, proteomics, and phosphoproteomics, we identified universal molecular mechanisms across distinct models of age-related and inherited retinal degenerations, characterized by impaired physiological resilience to stress. Here, we report that selective, targeted pharmacological inhibition of cyclic nucleotide phosphodiesterases (PDEs), which serve as critical regulatory nodes that modulate intracellular second messenger signaling pathways, stabilized the transcriptome, proteome, and phosphoproteome through downstream activation of protective mechanisms coupled with synergistic inhibition of degenerative processes. This therapeutic intervention enhanced resilience to acute and chronic forms of stress in the degenerating retina, thus preserving tissue structure and function across various models of age-related and inherited retinal disease. Taken together, these findings exemplify a systems pharmacology approach to drug discovery and development, revealing a new class of therapeutics with potential clinical utility in the treatment or prevention of the most common causes of blindness.
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Retinopatia Diabética , Degeneração Macular , Degeneração Retiniana , Retinose Pigmentar , Humanos , Retina/metabolismo , Degeneração Retiniana/metabolismo , Retinose Pigmentar/metabolismo , Degeneração Macular/patologia , Retinopatia Diabética/metabolismoRESUMO
Endothelial cell (EC) activation is a crucial determinant of retinal vascular inflammation associated with diabetic retinopathy (DR), a major microvascular complication of diabetes. We previously showed that, similar to abnormal biochemical factors, aberrant mechanical cues in the form of lysyl oxidase (LOX)-dependent subendothelial matrix stiffening also contribute significantly to retinal EC activation in diabetes. Yet, how LOX is itself regulated and precisely how it mechanically controls retinal EC activation in diabetes is poorly understood. Here, we show that high-glucose-induced LOX upregulation in human retinal ECs (HRECs) is mediated by proinflammatory receptor for advanced glycation end products (RAGE). HRECs treated with methylglyoxal (MGO), an active precursor to the advanced glycation end product (AGE) MG-H1, exhibited LOX upregulation that was blocked by a RAGE inhibitor, thus confirming the ability of RAGE to promote LOX expression. Crucially, as a downstream effector of RAGE, LOX was found to mediate both the proinflammatory and matrix remodeling effects of AGE/RAGE, primarily through its ability to crosslink or stiffen matrix. Finally, using decellularized HREC-derived matrices and a mouse model of diabetes, we demonstrate that LOX-dependent matrix stiffening feeds back to enhance RAGE, thereby achieving its autoregulation and proinflammatory effects. Collectively, these findings provide fresh mechanistic insights into the regulation and proinflammatory role of LOX-dependent mechanical cues in diabetes while simultaneously implicating LOX as an alternative (downstream) target to block AGE/RAGE signaling in DR. ARTICLE HIGHLIGHTS: We investigated the regulation and proinflammatory role of retinal endothelial lysyl oxidase (LOX) in diabetes. Findings reveal that LOX is upregulated by advanced glycation end products (AGE) and receptor for AGE (RAGE) and mediates AGE/RAGE-induced retinal endothelial cell activation and subendothelial matrix remodeling. We also show that LOX-dependent subendothelial matrix stiffening feeds back to enhance retinal endothelial RAGE. These findings implicate LOX as a key proinflammatory factor and an alternative (downstream) target to block AGE/RAGE signaling in diabetic retinopathy.
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Diabetes Mellitus , Retinopatia Diabética , Camundongos , Animais , Humanos , Retinopatia Diabética/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Proteína-Lisina 6-Oxidase/metabolismo , Retina/metabolismo , Endotélio/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Diabetes Mellitus/metabolismoRESUMO
Purpose: Limited research exists on the time course of long-term retinal and cerebral deficits in diabetic rodents. Previously, we examined short term (4-8 weeks) deficits in the Goto-Kakizaki (GK) rat model of Type II diabetes. Here, we investigated the long-term (1-8 months) temporal appearance of functional deficits (retinal, cognitive, and motor), retinal vascular pathology, and retinal dopamine levels in the GK rat. Methods: In GK rats and Wistar controls, retinal neuronal function (electroretinogram), cognitive function (Y-maze), and motor function (rotarod) were measured at 1, 2, 4, 6, and 8 months of age. In addition, we evaluated retinal vascular function (functional hyperemia) and glucose and insulin tolerance. Retinas from rats euthanized at ≥8 months were assessed for vascular pathology. Dopamine and DOPAC levels were measured via HPLC in retinas from rats euthanized at 1, 2, 8, and 12 months. Results: Goto-Kakizaki rats exhibited significant glucose intolerance beginning at 4 weeks and worsening over time (p < 0.001). GK rats also showed significant delays in flicker and oscillatory potential implicit times (p < 0.05 to p < 0.001) beginning at 1 month. Cognitive deficits were observed beginning at 6 months (p < 0.05), but no motor deficits. GK rats showed no deficits in functional hyperemia and no increase in acellular retinal capillaries. Dopamine levels were twice as high in GK vs. Wistar retinas at 1, 2, 8, and 12 months (p < 0.001). Conclusion: As shown previously, retinal deficits were detectable prior to cognitive deficits in GK rats. While retinal neuronal function was compromised, retinal vascular pathology was not observed, even at 12+ months. High endogenous levels of dopamine in the GK rat may be acting as an anti-angiogenic and providing protection against vascular pathology.
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AIMS/HYPOTHESIS: Accumulating evidence suggests that leucocytes play a critical role in diabetes-induced vascular lesions and other abnormalities that characterise the early stages of diabetic retinopathy. However, the role of monocytes has yet to be fully investigated; therefore, we used Ccr2-/- mice to study the role of CCR2+ inflammatory monocytes in the pathogenesis of diabetes-induced degeneration of retinal capillaries. METHODS: Experimental diabetes was induced in wild-type and Ccr2-/- mice using streptozotocin. After 2 months, superoxide levels, expression of inflammatory genes, leucostasis, leucocyte- and monocyte-mediated cytotoxicity against retinal endothelial cell death, retinal thickness and visual function were evaluated. Retinal capillary degeneration was determined after 8 months of diabetes. Flow cytometry of peripheral blood for differential expression of CCR2 in monocytes was assessed. RESULTS: In nondiabetic mice, CCR2 was highly expressed on monocytes, and Ccr2-/- mice lack CCR2+ monocytes in the peripheral blood. Diabetes-induced retinal superoxide, expression of proinflammatory genes Inos and Icam1, leucostasis and leucocyte-mediated cytotoxicity against retinal endothelial cells were inhibited in diabetic Ccr2-deficient mice and in chimeric mice lacking Ccr2 only from myeloid cells. In order to focus on monocytes, these cells were immuno-isolated after 2 months of diabetes, and they significantly increased monocyte-mediated endothelial cell cytotoxicity ex vivo. Monocytes from Ccr2-deficient mice caused significantly less endothelial cell death. The diabetes-induced retinal capillary degeneration was inhibited in Ccr2-/- mice and in chimeric mice lacking Ccr2 only from myeloid cells. CONCLUSIONS/INTERPRETATION: CCR2+ inflammatory monocytes contribute to the pathogenesis of early lesions of diabetic retinopathy.
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Diabetes Mellitus Experimental , Retinopatia Diabética , Degeneração Retiniana , Animais , Camundongos , Retinopatia Diabética/metabolismo , Monócitos/metabolismo , Células Endoteliais/metabolismo , Superóxidos/metabolismo , Degeneração Retiniana/metabolismo , Diabetes Mellitus Experimental/metabolismo , Camundongos Endogâmicos C57BL , Vasos Retinianos/patologia , Receptores CCR2/genética , Receptores CCR2/metabolismoRESUMO
SUMMARY STATEMENT: Diabetic human and murine retinas revealed pronounced microglial morphological activation and vascular abnormalities associated with inflammation. Pharmacological fibrinogen depletion using ancrod dampened microglial morphology alterations, resolved fibrinogen accumulation, rescued axonal integrity, and reduced inflammation in the diabetic murine retina.
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Ancrod , Retina , Animais , Receptor 1 de Quimiocina CX3C/genética , Fibrinogênio , Humanos , Inflamação/tratamento farmacológico , Camundongos , Microglia , Retina/fisiologiaRESUMO
AIMS/HYPOTHESIS: Induction of intercellular adhesion molecule-1 (ICAM-1) has been implicated in the development of macrovascular and microvascular diseases such as diabetic retinopathy. Lesions of diabetic retinopathy are unique to the retina but the reason for this is unclear, as all tissues are exposed to the same hyperglycaemic insult. We tested whether diabetes induces ICAM-1 on the luminal surface of endothelial cells to a greater extent in the retina than in other tissues and the role of vision itself in that induction. METHODS: Experimental diabetes was induced in C57Bl/6J, P23H opsin mutant and Gnat1-/- × Gnat2-/- double knockout mice using streptozotocin. The relative abundance of ICAM-1 on the luminal surface of endothelial cells in retina and other tissues was determined by conjugating anti-ICAM-1 antibodies to fluorescent microspheres (2 µm), injecting them intravenously and allowing them to circulate for 30 min. After transcardial perfusion, quantification of microspheres adherent to the endothelium in tissues throughout the body was carried out by fluorescent microscopy or flow cytometry. Mice injected with lipopolysaccharide (LPS) were used as positive controls. The difference in leucostasis between retinal and non-retinal vasculature was evaluated. RESULTS: Diabetes significantly increased ICAM-1-mediated adherence of microspheres to retinal microvessels by almost threefold, independent of sex. In contrast, diabetes had a much smaller effect on endothelial ICAM-1 in other tissues, and more tissues showed a significant induction of endothelial ICAM-1 with LPS than with diabetes. The diabetes-induced increase in endothelial ICAM-1 in retinal vasculature was inhibited by blocking phototransduction in photoreceptor cells. Diabetes significantly increased leucostasis in the retina by threefold compared with a non-ocular tissue (cremaster). CONCLUSIONS/INTERPRETATION: The diabetes-induced upregulation of ICAM-1 on the luminal surface of the vascular endothelium varies considerably among tissues and is highest in the retina. Induction of ICAM-1 on retinal vascular endothelial cells in diabetes is influenced by vision-related processes in photoreceptor cells. The unique presence of photoreceptors in the retina might contribute to the greater susceptibility of this tissue to vascular disease in diabetes.
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Diabetes Mellitus Experimental , Retinopatia Diabética , Molécula 1 de Adesão Intercelular/metabolismo , Animais , Células Endoteliais , Lipopolissacarídeos/efeitos adversos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Opsinas , EstreptozocinaRESUMO
Targeted drug delivery to disease-associated activated neutrophils can provide novel therapeutic opportunities while avoiding systemic effects on immune functions. We created a nanomedicine platform that uniquely utilizes an α1-antitrypsin-derived peptide to confer binding specificity to neutrophil elastase on activated neutrophils. Surface decoration with this peptide enabled specific anchorage of nanoparticles to activated neutrophils and platelet-neutrophil aggregates, in vitro and in vivo. Nanoparticle delivery of a model drug, hydroxychloroquine, demonstrated significant reduction of neutrophil activities in vitro and a therapeutic effect on murine venous thrombosis in vivo. This innovative approach of cell-specific and activation-state-specific targeting can be applied to several neutrophil-driven pathologies.
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Elastase de Leucócito , Deficiência de alfa 1-Antitripsina , Animais , Humanos , Hidroxicloroquina/farmacologia , Elastase de Leucócito/metabolismo , Camundongos , Nanomedicina , NeutrófilosRESUMO
PURPOSE: To determine if treatment with a photobiomodulation (PBM) device results in greater improvement in central subfield thickness (CST) than placebo in eyes with center-involved diabetic macular edema (CI-DME) and good vision. DESIGN: Phase 2 randomized clinical trial. PARTICIPANTS: Participants had CI-DME and visual acuity (VA) 20/25 or better in the study eye and were recruited from 23 clinical sites in the United States. METHODS: One eye of each participant was randomly assigned 1:1 to a 670-nm light-emitting PBM eye patch or an identical device emitting broad-spectrum white light at low power. Treatment was applied for 90 seconds twice daily for 4 months. MAIN OUTCOME MEASURES: Change in CST on spectral-domain OCT at 4 months. RESULTS: From April 2019 to February 2020, 135 adults were randomly assigned to either PBM (n = 69) or placebo (n = 66); median age was 62 years, 37% were women, and 82% were White. The median device compliance was 92% with PBM and 95% with placebo. OCT CST increased from baseline to 4 months by a mean (SD) of 13 (53) µm in PBM eyes and 15 (57) µm in placebo eyes, with the mean difference (95% confidence interval [CI]) being -2 (-20 to 16) µm (P = 0.84). CI-DME, based on DRCR Retina Network sex- and machine-based thresholds, was present in 61 (90%) PBM eyes and 57 (86%) placebo eyes at 4 months (adjusted odds ratio [95% CI] = 1.30 (0.44-3.83); P = 0.63). VA decreased by a mean (SD) of -0.2 (5.5) letters and -0.6 (4.6) letters in the PBM and placebo groups, respectively (difference [95% CI] = 0.4 (-1.3 to 2.0) letters; P = 0.64). There were 8 adverse events possibly related to the PBM device and 2 adverse events possibly related to the placebo device. None were serious. CONCLUSIONS: PBM as given in this study, although safe and well-tolerated, was not found to be effective for the treatment of CI-DME in eyes with good vision.
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Diabetes Mellitus , Retinopatia Diabética , Terapia com Luz de Baixa Intensidade , Edema Macular , Adulto , Inibidores da Angiogênese/uso terapêutico , Ensaios Clínicos Fase II como Assunto , Diabetes Mellitus/tratamento farmacológico , Retinopatia Diabética/complicações , Retinopatia Diabética/diagnóstico , Retinopatia Diabética/terapia , Feminino , Humanos , Edema Macular/tratamento farmacológico , Edema Macular/terapia , Masculino , Pessoa de Meia-Idade , Ensaios Clínicos Controlados Aleatórios como Assunto , Tomografia de Coerência Óptica/métodos , Acuidade VisualRESUMO
Purpose: Previous studies indicate that leukocytes, notably neutrophils, play a causal role in the capillary degeneration observed in diabetic retinopathy (DR), however, the mechanism by which they cause such degeneration is unknown. Neutrophil elastase (NE) is a protease released by neutrophils which participates in a variety of inflammatory diseases. In the present work, we investigated the potential involvement of NE in the development of early DR. Methods: Experimental diabetes was induced in NE-deficient mice (Elane-/-), in mice treated daily with the NE inhibitor, sivelestat, and in mice overexpressing human alpha-1 antitrypsin (hAAT+). Mice were assessed for diabetes-induced retinal superoxide generation, inflammation, leukostasis, and capillary degeneration. Results: In mice diabetic for 2 months, deletion of NE or selective inhibition of NE inhibited diabetes-induced retinal superoxide levels and inflammation, and inhibited leukocyte-mediated cytotoxicity of retinal endothelial cells. In mice diabetic for 8 months, genetic deletion of NE significantly inhibited diabetes-induced retinal capillary degeneration. Conclusions: These results suggest that a protease released from neutrophils contributes to the development of DR, and that blocking NE activity could be a novel therapy to inhibit DR.
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Diabetes Mellitus Experimental/complicações , Retinopatia Diabética/metabolismo , Neutrófilos/enzimologia , Peptídeo Hidrolases/sangue , Retina/metabolismo , Animais , Retinopatia Diabética/diagnóstico , Retinopatia Diabética/etiologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Retina/diagnóstico por imagemRESUMO
We compared monotherapies and combinations of therapies that regulate G-protein-coupled receptors (GPCRs) with respect to their abilities to inhibit early stages of diabetic retinopathy (DR) in streptozotocin-diabetic mice. Metoprolol (MTP; 0.04-1.0 mg/kg b.wt./day), bromocriptine (BRM; 0.01-0.1 mg/kg b.wt./day), doxazosin (DOX; 0.01-1.0 mg/kg b.wt./day), or tamsulosin (TAM; 0.05-0.25 mg/kg b.wt./day) were injected individually daily for 2 months in dose-response studies to assess their effects on the diabetes-induced increases in retinal superoxide and leukocyte-mediated cytotoxicity against vascular endothelial cells, both of which abnormalities have been implicated in the development of DR. Each of the individual drugs inhibited the diabetes-induced increase in retinal superoxide at the higher concentrations tested, but the inhibition was lost at lower doses. To determine whether combination therapies had superior effects over individual drugs, we intentionally selected for each drug a low dose that had little or no effect on the diabetes-induced retinal superoxide for use separately or in combinations in 8-month studies of retinal function, vascular permeability, and capillary degeneration in diabetes. At the low doses used, combinations of the drugs generally were more effective than individual drugs, but the low-dose MTP alone totally inhibited diabetes-induced reduction in a vision task, BRM or DOX alone totally inhibited the vascular permeability defect, and DOX alone totally inhibited diabetes-induced degeneration of retinal capillaries. Although low-dose MTP, BRM, DOX, or TAM individually had beneficial effects on some endpoints, combination of the therapies better inhibited the spectrum of DR lesions evaluated. SIGNIFICANCE STATEMENT: The pathogenesis of early stages of diabetic retinopathy remains incompletely understood, but multiple different cell types are believed to be involved in the pathogenic process. We have compared the effects of monotherapies to those of combinations of drugs that regulate GPCR signaling pathways with respect to their relative abilities to inhibit the development of early diabetic retinopathy.
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Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/metabolismo , Hipoglicemiantes/administração & dosagem , Receptores Adrenérgicos/metabolismo , Receptores Dopaminérgicos/metabolismo , Receptores de Serotonina/metabolismo , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/patologia , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/metabolismo , Vasos Retinianos/patologiaRESUMO
This study tested the hypothesis that diabetes promotes a greater than normal cytosolic calcium level in rod cells that activates a Ca2+-sensitive protease, calpain, resulting in oxidative stress and inflammation, two pathogenic factors of early diabetic retinopathy. Nondiabetic and 2-month diabetic C57Bl/6J and calpain1 knockout (Capn1-/-) mice were studied; subgroups were treated with a calpain inhibitor (CI). Ca2+ content was measured in photoreceptors using Fura-2. Retinal calpain expression was studied by quantitative RT-PCR and immunohistochemistry. Superoxide and expression of inflammatory proteins were measured using published methods. Proteomic analysis was conducted on photoreceptors isolated from untreated diabetic mice or treated daily with CI for 2 months. Cytosolic Ca2+ content was increased twofold in photoreceptors of diabetic mice as compared with nondiabetic mice. Capn1 expression increased fivefold in photoreceptor outer segments of diabetic mice. Pharmacologic inhibition or genetic deletion of Capn1 significantly suppressed diabetes-induced oxidative stress and expression of proinflammatory proteins in retina. Proteomics identified a protein (WW domain-containing oxidoreductase [WWOX]) whose expression was significantly increased in photoreceptors from mice diabetic for 2 months and was inhibited with CI. Knockdown of Wwox using specific siRNA in vitro inhibited increase in superoxide caused by the high glucose. These results suggest that reducing Ca2+ accumulation, suppressing calpain activation, and/or reducing Wwox up-regulation are novel targets for treating early diabetic retinopathy.
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Cálcio/metabolismo , Calpaína/metabolismo , Retinopatia Diabética/patologia , Inflamação/patologia , Estresse Oxidativo , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patologia , Animais , Calpaína/genética , Linhagem Celular , Retinopatia Diabética/complicações , Retinopatia Diabética/genética , Retinopatia Diabética/fisiopatologia , Ativação Enzimática/efeitos dos fármacos , Deleção de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Glicoproteínas/farmacologia , Inflamação/complicações , Inflamação/genética , Inflamação/fisiopatologia , Molécula 1 de Adesão Intercelular/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteoma/metabolismo , Retina/patologia , Índice de Gravidade de Doença , Superóxidos/metabolismo , Regulação para Cima/efeitos dos fármacos , Visão Ocular/efeitos dos fármacos , Oxidorredutase com Domínios WW/metabolismoRESUMO
While the administration of anti-CD154 mAbs in mice validated the CD40-CD154 pathway as a target against inflammatory disorders, this approach caused thromboembolism in humans (unrelated to CD40 inhibition) and is expected to predispose to opportunistic infections. There is a need for alternative approaches to inhibit CD40 that avoid these complications. CD40 signals through TRAF2,3 and TRAF6-binding sites. Given that CD40-TRAF6 is the pathway that stimulates responses key for cell-mediated immunity against opportunistic pathogens, we examined the effects of pharmacologic inhibition of CD40-TRAF2,3 signaling. We used a model of ischemia/reperfusion (I/R)-induced retinopathy, a CD40-driven inflammatory disorder. Intravitreal administration of a cell-penetrating CD40-TRAF2,3 blocking peptide impaired ICAM-1 upregulation in retinal endothelial cells and CXCL1 upregulation in endothelial and Müller cells. The peptide reduced leukocyte infiltration, upregulation of NOS2/COX-2/TNF-α/IL-1ß, and ameliorated neuronal loss, effects that mimic those observed after I/R in Cd40-/- mice. While a cell-penetrating CD40-TRAF6 blocking peptide also diminished I/R-induced inflammation, this peptide (but not the CD40-TRAF2,3 blocking peptide) impaired control of the opportunistic pathogen Toxoplasma gondii in the retina. Thus, inhibition of the CD40-TRAF2,3 pathway is a novel and potent approach to reduce CD40-induced inflammation, while likely diminishing the risk of opportunistic infections that would otherwise accompany CD40 inhibition.
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Antígenos CD40/efeitos dos fármacos , Inflamação/tratamento farmacológico , Neurônios/efeitos dos fármacos , Peptídeos/farmacologia , Fator 2 Associado a Receptor de TNF/metabolismo , Animais , Antígenos CD40/genética , Antígenos CD40/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Humanos , Inflamação/metabolismo , Isquemia/tratamento farmacológico , Isquemia/metabolismo , Masculino , Camundongos , Neurônios/citologia , Reperfusão/métodos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator 2 Associado a Receptor de TNF/efeitos dos fármacosRESUMO
PURPOSE: The phosphodiesterase inhibitor sildenafil is a promising treatment for neurodegenerative disease, but it can cause oxidative stress in photoreceptors ex vivo and degrade visual performance in humans. Here, we test the hypotheses that in wildtype mice sildenafil causes i) wide-spread photoreceptor oxidative stress in vivo that is linked with ii) impaired vision. METHODS: In dark or light-adapted C57BL/6 mice ± sildenafil treatment, the presence of oxidative stress was evaluated in retina laminae in vivo by QUEnch-assiSTed (QUEST) magnetic resonance imaging, in the subretinal space in vivo by QUEST optical coherence tomography, and in freshly excised retina by a dichlorofluorescein assay. Visual performance indices were also evaluated by QUEST optokinetic tracking. RESULTS: In light-adapted mice, 1 hr post-sildenafil administration, oxidative stress was most evident in the superior peripheral outer retina on both in vivo and ex vivo examinations; little evidence was noted for central retina oxidative stress in vivo and ex vivo. In dark-adapted mice 1 hr after sildenafil, no evidence for outer retina oxidative stress was found in vivo. Evidence for sildenafil-induced central retina rod cGMP accumulation was suggested as a panretinally thinner, dark-like subretinal space thickness in light-adapted mice at 1 hr but not 5 hr post-sildenafil. Cone-based visual performance was impaired by 5 hr post-sildenafil and not corrected with anti-oxidants; vision was normal at 1 hr and 24 hr post-sildenafil. CONCLUSIONS: The sildenafil-induced spatiotemporal pattern of oxidative stress in photoreceptors dominated by rods was unrelated to impairment of cone-based visual performance in wildtype mice.
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Estresse Oxidativo , Inibidores de Fosfodiesterase/farmacologia , Células Fotorreceptoras/efeitos dos fármacos , Citrato de Sildenafila/farmacologia , Visão Ocular , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Fotorreceptoras/metabolismoRESUMO
O-GlcNAcylation is a reversible post-translational protein modification that regulates fundamental cellular processes including immune responses and autoimmunity. Previously, we showed that hyperglycemia increases O-GlcNAcylation of the transcription factor, nuclear factor kappaB c-Rel at serine residue 350 and enhances the transcription of the c-Rel-dependent proautoimmune cytokines interleukin-2, interferon gamma and granulocyte macrophage colony stimulating factor in T cells. c-Rel also plays a critical role in the transcriptional regulation of forkhead box P3 (FOXP3)-the master transcription factor that governs development and function of Treg cells. Here we show that the regulatory effect of c-Rel O-GlcNAcylation is gene-dependent, and in contrast to its role in enhancing the expression of proautoimmune cytokines, it suppresses the expression of FOXP3. Hyperglycemia-induced O-GlcNAcylation-dependent suppression of FOXP3 expression was found in vivo in two mouse models of autoimmune diabetes; streptozotocin-induced diabetes and spontaneous diabetes in nonobese diabetic mice. Mechanistically, we show that both hyperglycemia-induced and chemically enhanced cellular O-GlcNAcylation decreases c-Rel binding at the FOXP3 promoter and negatively regulates FOXP3 expression. Mutation of the O-GlcNAcylation site in c-Rel, (serine 350 to alanine), augments T cell receptor-induced FOXP3 expression and resists the O-GlcNAcylation-dependent repression of FOXP3 expression. This study reveals c-Rel S350 O-GlcNAcylation as a novel molecular mechanism inversely regulating immunosuppressive FOXP3 expression and proautoimmune gene expression in autoimmune diabetes with potential therapeutic implications.
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Diabetes Mellitus Experimental , Fatores de Transcrição Forkhead , Proteínas Proto-Oncogênicas c-rel , Animais , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Camundongos , Proteínas Proto-Oncogênicas c-rel/genética , Proteínas Proto-Oncogênicas c-rel/metabolismo , Proteínas Proto-Oncogênicas c-rel/farmacologia , Linfócitos T ReguladoresRESUMO
Photoreceptors have high energy demands and a high density of mitochondria that produce ATP through oxidative phosphorylation (OXPHOS) of fuel substrates. Although glucose is the major fuel for CNS brain neurons, in photoreceptors (also CNS), most glucose is not metabolized through OXPHOS but is instead metabolized into lactate by aerobic glycolysis. The major fuel sources for photoreceptor mitochondria remained unclear for almost six decades. Similar to other tissues (like heart and skeletal muscle) with high metabolic rates, photoreceptors were recently found to metabolize fatty acids (palmitate) through OXPHOS. Disruption of lipid entry into photoreceptors leads to extracellular lipid accumulation, suppressed glucose transporter expression, and a duel lipid/glucose fuel shortage. Modulation of lipid metabolism helps restore photoreceptor function. However, further elucidation of the types of lipids used as retinal energy sources, the metabolic interaction with other fuel pathways, as well as the cross-talk among retinal cells to provide energy to photoreceptors is not fully understood. In this review, we will focus on the current understanding of photoreceptor energy demand and sources, and potential future investigations of photoreceptor metabolism.
Assuntos
Células FotorreceptorasRESUMO
It is still a challenge to develop gene replacement therapy for retinal disorders caused by mutations in large genes, such as Stargardt disease (STGD). STGD is caused by mutations in ABCA4 gene. Previously, we have developed an effective non-viral gene therapy using self-assembled nanoparticles of a multifunctional pH-sensitive amino lipid ECO and a therapeutic ABCA4 plasmid containing rhodopsin promoter (pRHO-ABCA4). In this study, we modified the ABCA4 plasmid with simian virus 40 enhancer (SV40, pRHO-ABCA4-SV40) for enhanced gene expression. We also prepared and assessed the formulations of ECO/pDNA nanoparticles using sucrose or sorbitol as a stablilizer to develop consistent and stable formulations. Results demonstrated that ECO formed stable nanoparticles with pRHO-ABCA4-SV40 in the presence of sucrose, but not with sorbitol. The transfection efficiency in vitro increased significantly after introduction of SV40 enhancer for plasmid pCMV-ABCA4-SV40 with a CMV promoter. Sucrose didn't affect the transfection efficiency, while sorbitol resulted in a fluctuation of the in vitro transfection efficiency. Subretinal gene therapy in Abca4-/- mice using ECO/pRHO-ABCA4 and ECO/pRHO-ABCA4-SV40 nanoparticles induced 36% and 29% reduction in A2E accumulation respectively. Therefore, the ECO/pABCA4 based nanoparticles are promising for non-viral gene therapy for Stargardt disease and can be expended for applications in a variety of visual dystrophies with mutated large genes.
Assuntos
Nanopartículas , Vírus 40 dos Símios , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Terapia Genética , Camundongos , Mutação , Doença de StargardtRESUMO
Diabetic retinopathy (DR) is a leading cause of blindness. It has long been regarded as vascular disease, but work in the past years has shown abnormalities also in the neural retina. Unfortunately, research on the vascular and neural abnormalities have remained largely separate, instead of being integrated into a comprehensive view of DR that includes both the neural and vascular components. Recent evidence suggests that the most predominant neural cell in the retina (photoreceptors) and the adjacent retinal pigment epithelium (RPE) play an important role in the development of vascular lesions characteristic of DR. This review summarizes evidence that the outer retina is altered in diabetes, and that photoreceptors and RPE contribute to retinal vascular alterations in the early stages of the retinopathy. The possible molecular mechanisms by which cells of the outer retina might contribute to retinal vascular damage in diabetes also are discussed. Diabetes-induced alterations in the outer retina represent a novel therapeutic target to inhibit DR.
Assuntos
Diabetes Mellitus , Retinopatia Diabética , Humanos , Células Fotorreceptoras , Retina , Epitélio Pigmentado da RetinaRESUMO
PURPOSE: Female mice have been found to be resistant to streptozotocin (STZ)-induced diabetes, and pre-clinical research related to diabetic complications commonly omits females. The purpose of this study was to develop a method to induce diabetes in female mice, and to determine if retinas of diabetic female mice develop molecular changes and histopathological abnormalities comparable to those which develop in male diabetic mice. METHODS: To induce diabetes, animals of both sexes received daily intraperitoneal (i.p.) injection of STZ for 5 consecutive days at 55 mg/kg BW (a dose that is known to induce diabetes in male mice) or for females, 75 mg/kg BW of STZ. Retinal abnormalities that have been implicated in the development of the retinopathy (superoxide generation and expression of inflammatory proteins, iNOS and ICAM-1) were evaluated at 2 months of diabetes, and retinal capillary degeneration was evaluated at 8 months of diabetes. RESULTS: Daily i.p. injection of STZ for 5 consecutive days at a concentration of 55 mg/kg BW was sufficient to induce diabetes in 100% of male mice, but only 33% of female mice. However, females did become hyperglycemic when the dose of STZ administered was increased to 75 mg/kg BW. The resulting STZ-induced hyperglycemia in female and male mice was sustained for at least 8 months. After induction of the diabetes, both sexes responded similarly with respect to the oxidative stress, expression of iNOS, and degeneration of retinal capillaries, but differed in the limited population evaluated with respect to expression of ICAM-1. CONCLUSIONS: The resistance of female mice to STZ-induced diabetes can be overcome by increasing the dose of STZ used. Female mice can, and should, be included in pre-clinical studies of diabetes and its complications.