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Idiopathic pulmonary fibrosis (IPF) is an aggressive interstitial lung disease with a high mortality rate. Putative drug targets in IPF have failed to translate into effective therapies at the clinical level. We identify TRAF2- and NCK-interacting kinase (TNIK) as an anti-fibrotic target using a predictive artificial intelligence (AI) approach. Using AI-driven methodology, we generated INS018_055, a small-molecule TNIK inhibitor, which exhibits desirable drug-like properties and anti-fibrotic activity across different organs in vivo through oral, inhaled or topical administration. INS018_055 possesses anti-inflammatory effects in addition to its anti-fibrotic profile, validated in multiple in vivo studies. Its safety and tolerability as well as pharmacokinetics were validated in a randomized, double-blinded, placebo-controlled phase I clinical trial (NCT05154240) involving 78 healthy participants. A separate phase I trial in China, CTR20221542, also demonstrated comparable safety and pharmacokinetic profiles. This work was completed in roughly 18 months from target discovery to preclinical candidate nomination and demonstrates the capabilities of our generative AI-driven drug-discovery pipeline.
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Heparan-α-glucosaminide N-acetyltransferase (HGSNAT) participates in lysosomal degradation of heparan sulfate. Mutations in the gene encoding this enzyme cause mucopolysaccharidosis IIIC (MPS IIIC) or Sanfilippo syndrome type C. MPS IIIC patients exhibit progressive neurodegeneration, leading to dementia and death in early adulthood. Currently there is no approved treatment for MPS IIIC. Incidences of non-syndromic retinitis pigmentosa and early signs of night blindness are reported in some MPS IIIC patients, however the majority of ocular phenotypes are not well characterized. The goal of this study was to investigate retinal degeneration phenotype in the Hgsnat knockout mouse model of MPS IIIC and a cadaveric human MPS IIIC eye. Cone and rod photoreceptors in the eyes of homozygous 6-month-old Hgsnat knockout mice and their wild-type counterparts were analyzed using cone arrestin, S-opsin, M-opsin and rhodopsin antibodies. Histological observation was performed on the eye from a 35-year-old MPS IIIC donor. We observed a nearly 50% reduction in the rod photoreceptors density in the Hgsnat knockout mice compared to the littermate wild-type controls. Cone photoreceptor density was unaltered at this age. Severe retinal degeneration was also observed in the MPS IIIC donor eye. To our knowledge, this is the first report characterizing ocular phenotypes arising from deleterious variants in the Hgsnat gene associated with MPS IIIC clinical phenotype. Our findings indicate retinal manifestations may be present even before behavioral manifestations. Thus, we speculate that ophthalmological evaluations could be used as diagnostic indicators of early disease, progression, and end-point evaluation for future MPS IIIC therapies.
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Mucopolisacaridosis III , Degeneración Retiniana , Retinitis Pigmentosa , Animales , Ratones , Humanos , Adulto , Lactante , Mucopolisacaridosis III/genética , Mucopolisacaridosis III/diagnóstico , Mucopolisacaridosis III/patología , Degeneración Retiniana/genética , Mutación , Ratones Noqueados , Acetiltransferasas/genéticaRESUMEN
BACKGROUND: Ramucirumab is indicated for patients with advanced hepatocellular carcinoma (HCC) and α-fetoprotein (AFP) ≥400 ng/mL following sorafenib. Here, we prospectively studied ramucirumab following non-sorafenib systemic therapies. MATERIALS AND METHODS: This open-label, non-comparative cohort of REACH-2 enrolled patients with advanced HCC, Child-Pugh class-A liver disease, and AFP ≥400 ng/mL who had received 1-2 lines of therapy, excluding sorafenib or chemotherapy. Ramucirumab was administered 8 mg/kg intravenously Q2W. The primary endpoint was safety. Secondary endpoints were overall survival, progression-free survival, objective response rate (RECIST v1.1), time to progression, pharmacokinetics, and patient-reported outcomes. Final analysis occurred after all enrolled patients completed ≥3 treatment cycles or discontinued treatment. RESULTS: Between April 27, 2018, and March 29, 2021, 47 patients were treated at 21 investigative sites in Asia, Europe, and USA. The most frequently reported grade ≥3 adverse events, regardless of causality, were hypertension (11%), proteinuria (6%), hyponatremia (6%), and AST increased (6%). Two patients died from adverse events (myocardial infarction and upper gastrointestinal hemorrhage), deemed related to treatment. Median progression-free survival, time to progression, and overall survival were 1.7 months, 2.8 months, and 8.7 months, respectively. The objective response rate was 10.6% with a median duration response of 8.3 months. Median time to deterioration in FHSI-8 total score was 4.4 months. CONCLUSION: Ramucirumab demonstrated consistent and meaningful clinical activity with no new safety signals following non-sorafenib therapies in patients with advanced HCC and AFP ≥400 ng/mL. This represents one of the first sequencing studies for patients with advanced HCC not treated with sorafenib.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Sorafenib/uso terapéutico , Carcinoma Hepatocelular/tratamiento farmacológico , alfa-Fetoproteínas , Neoplasias Hepáticas/tratamiento farmacológico , Europa (Continente)RESUMEN
Circadian clocks in the mammalian retina regulate a diverse range of retinal functions that allow the retina to adapt to the light-dark cycle. Emerging evidence suggests a link between the circadian clock and retinopathies though the causality has not been established. Here we report that clock genes are expressed in the mouse embryonic retina, and the embryonic retina requires light cues to maintain robust circadian expression of the core clock gene, Bmal1. Deletion of Bmal1 and Per2 from the retinal neurons results in retinal angiogenic defects similar to when animals are maintained under constant light conditions. Using two different models to assess pathological neovascularization, we show that neuronal Bmal1 deletion reduces neovascularization with reduced vascular leakage, suggesting that a dysregulated circadian clock primarily drives neovascularization. Chromatin immunoprecipitation sequencing analysis suggests that semaphorin signaling is the dominant pathway regulated by Bmal1. Our data indicate that therapeutic silencing of the retinal clock could be a common approach for the treatment of certain retinopathies like diabetic retinopathy and retinopathy of prematurity.
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Relojes Circadianos , Ritmo Circadiano , Animales , Relojes Circadianos/genética , Ritmo Circadiano/genética , Mamíferos , Ratones , Neovascularización Patológica/metabolismo , Fotoperiodo , Retina/metabolismoRESUMEN
PURPOSE: The purpose of this study is to assess for histopathological changes within the retina and the choroid and determine the long-term sequelae of the SARS-CoV-2 infection. METHODS: Eyes from seven COVID-19-positive and six similar age-matched control donors with a negative test for SARS-CoV-2 were assessed. Globes were evaluated ex vivo with macroscopic, SLO and OCT imaging. Macula and peripheral regions were processed for Epon embedding and immunocytochemistry. RESULTS: Fundus analysis shows hemorrhagic spots and increased vitreous debris in several of the COVID-19 eyes compared to the controls. OCT-based measurements indicated an increased trend in retinal thickness in the COVID-19 eyes; however, the difference was not statistically significant. Histology of the retina showed presence of hemorrhages and central cystoid degeneration in several of the donors. Whole mount analysis of the retina labeled with markers showed changes in retinal microvasculature, increased inflammation, and gliosis in the COVID-19 eyes compared to the controls. The choroidal vasculature displayed localized changes in density and signs of increased inflammation in the COVID-19 samples. CONCLUSIONS: In situ analysis of the retinal tissue suggests that there are severe subclinical abnormalities that could be detected in the COVID-19 eyes. This study provides a rationale for evaluating the ocular physiology of patients that have recovered from COVID-19 infections to further understand the long-term effects caused by this virus.
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COVID-19 , Mácula Lútea , COVID-19/complicaciones , Coroides/patología , Gliosis/diagnóstico , Gliosis/patología , Humanos , Inflamación/diagnóstico , Inflamación/patología , Retina , SARS-CoV-2 , Tomografía de Coherencia ÓpticaRESUMEN
Oxygen supplementation is necessary to prevent mortality in severely premature infants. However, the supraphysiological concentration of oxygen utilized in these infants simultaneously creates retinovascular growth attenuation and vasoobliteration that induces the retinopathy of prematurity. Here, we report that hyperoxia regulates the cell cycle and retinal endothelial cell proliferation in a previously unknown Myc-dependent manner, which contributes to oxygen-induced retinopathy.
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Sleep dysfunction has been identified in the pathophysiology of Alzheimer's disease (AD); however, the role and mechanism of circadian rhythm dysfunction is less well understood. In a well-characterized cohort of patients with AD at the mild cognitive impairment stage (MCI-AD), we identify that circadian rhythm irregularities were accompanied by altered humoral immune responses detected in both the cerebrospinal fluid and plasma as well as alterations of cerebrospinal fluid biomarkers of neurodegeneration. On the other hand, sleep disruption was more so associated with abnormalities in circulating markers of immunity and inflammation and decrements in cognition.
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Enfermedad de Alzheimer/fisiopatología , Ritmo Circadiano/fisiología , Cognición/fisiología , Disfunción Cognitiva/fisiopatología , Sueño/fisiología , Anciano , Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/líquido cefalorraquídeo , Péptidos beta-Amiloides/sangre , Péptidos beta-Amiloides/líquido cefalorraquídeo , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Disfunción Cognitiva/sangre , Disfunción Cognitiva/líquido cefalorraquídeo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Trastornos del Sueño-Vigilia/complicaciones , Proteínas tau/sangre , Proteínas tau/líquido cefalorraquídeoRESUMEN
PURPOSE: To assess for histopathological changes within the retina and the choroid and determine the long-term sequelae of the SARS-CoV-2 infection. DESIGN: Comparative analysis of human eyes. SUBJECTS: Eleven donor eyes from COVID-19 positive donors and similar age-matched donor eyes from patients with a negative test for SARS-CoV-2 were assessed. METHODS: Globes were evaluated ex-vivo with macroscopic, SLO and OCT imaging. Macula and peripheral regions were processed for epon-embedding and immunocytochemistry. MAIN OUTCOME MEASURES: Retinal thickness and histopathology, detection of SARS-CoV-2 Spike protein, changes in vascular density, gliosis, and degree of inflammation. RESULTS: Fundus analysis shows hemorrhagic spots and increased vitreous debris in several of the COVID-19 eyes compared to the control. OCT based measurements indicated an increased trend in retinal thickness in the COVID-19 eyes, however the difference was not statistically significant. Histology of the retina showed presence of hemorrhages and central cystoid degeneration in several of the donors. Whole mount analysis of the retina labeled with markers showed changes in retinal microvasculature, increased inflammation, and gliosis in the COVID-19 eyes compared to the controls. The choroidal vasculature displayed localized changes in density and signs of increased inflammation in the COVID-19 samples. CONCLUSIONS: In situ analysis of the retinal tissue suggested that there are severe subclinical abnormalities that could be detected in the COVID-19 eyes. This study provides a rationale for evaluating the ocular physiology of patients that have recovered from COVID-19 infections to further understand the long-term effects caused by this virus.
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PURPOSE: SEQUOIA compared efficacy and safety of adding pegilodecakin (PEG), a pegylated recombinant human interleukin (IL)-10, with folinic acid, fluorouracil, and oxaliplatin (FOLFOX) in patients following progression on first-line gemcitabine-containing therapy with metastatic pancreatic ductal adenocarcinoma (PDAC). PATIENTS AND METHODS: SEQUOIA, a randomized, global phase III study, compared FOLFOX with PEG + FOLFOX as second line in gemcitabine-refractory PDAC. Patients were randomly assigned 1:1 (PEG + FOLFOX:FOLFOX) and stratified by prior gemcitabine and region. Eligible patients had only one prior gemcitabine-containing treatment. Primary end point was overall survival (OS). Secondary end points included progression-free survival (PFS), response evaluation per Response Evaluation Criteria in Solid Tumor (RECIST) 1.1, and safety. Exploratory analyses included biomarkers related to immune activation. RESULTS: Between March 1, 2017, and September 9, 2019, 567 patients were randomly assigned PEG + FOLFOX (n = 283) or FOLFOX (n = 284). Most (94.7%) patients received prior gemcitabine plus nab paclitaxel. OS was similar comparing PEG + FOLFOX versus FOLFOX (median: 5.8 v 6.3 months; hazard ratio = 1.045; 95% CI, 0.863 to 1.265). Also, PFS (median 2.1 v 2.1 months; hazard ratio = 0.981; 95% CI, 0.808 to 1.190) and objective response rate (4.6% v 5.6%) were similar between the treatment arms. Most common (≥ 35%) treatment-emergent adverse events in PEG + FOLFOX versus FOLFOX were thrombocytopenia (55% v 20%), anemia (40% v 16%), fatigue (61% v 45%), neutropenia (39% v 28%), abdominal pain (37% v 29%), nausea (45% v 41%), neuropathy (37% v 38%), and decreased appetite (35% v 31%). Exploratory analyses revealed increases in total IL-18, interferon (IFN)-γ, and granzyme B and decreases in transforming growth factor (TGF)-ß with the addition of PEG. CONCLUSION: PEG added to FOLFOX did not improve efficacy in advanced gemcitabine-refractory PDAC. Safety findings were consistent as previously observed from PEG with chemotherapy; toxicity was manageable and tolerable. Exploratory pharmacodynamic results were consistent with immunostimulatory signals of the IL-10R pathway.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Carcinoma Ductal Pancreático/patología , Desoxicitidina/uso terapéutico , Progresión de la Enfermedad , Resistencia a Antineoplásicos , Fatiga/inducido químicamente , Femenino , Fluorouracilo/administración & dosificación , Fluorouracilo/efectos adversos , Humanos , Interleucina-10/administración & dosificación , Interleucina-10/efectos adversos , Estimación de Kaplan-Meier , Leucovorina/administración & dosificación , Leucovorina/efectos adversos , Masculino , Persona de Mediana Edad , Evaluación de Resultado en la Atención de Salud/métodos , Evaluación de Resultado en la Atención de Salud/estadística & datos numéricos , Oxaliplatino/administración & dosificación , Oxaliplatino/efectos adversos , Neoplasias Pancreáticas/patología , Polietilenglicoles/administración & dosificación , Polietilenglicoles/efectos adversos , Trombocitopenia/inducido químicamente , GemcitabinaRESUMEN
Background Treatment options for pancreatic ductal adenocarcinoma (PDAC) are limited and checkpoint blockade inhibitors have been disappointing in this disease. Pegilodecakin has demonstrated single agent anti-tumor activity in immune-sensitive tumors. Phase 1 and preclinical data indicate synergy of pegilodecakin with 5-FU and platins. We assessed the safety and activity of pegilodecakin+FOLFOX in patients with PDAC. Methods IVY (NCT02009449) was an open-label phase 1b trial in the United States. Here we report on all enrolled patients from cohort C. Heavily pretreated patients were treated with pegilodecakin (self-administered subcutaneously daily at 2.5, 5, or 10 µg/kg) + 5-flurouracil/leucovorin/oxaliplatin (FOLFOX), dosed per manufacturers prescribing information, until tumor progression. Eligible patients had measurable disease per immune-related response criteria (irRC), were ≥ 18 years of age, and had ECOG performance status of 0 or 1. Patients were evaluated for primary(safety) and secondary (tumor response per irRC) endpoints. Results From 5 August 2014-12 July 2016, 39 patients enrolled in cohort C. All patients were evaluable for safety. In this advanced population, regimen had manageable toxicities with no immune-related adverse events (irAEs) greater than grade 1. The most common grade 3/4/5 TEAEs were thrombocytopenia (21[53.8%] of 39) and anemia (17[43.6%] of 39). In evaluable PDAC patients, the best overall response of pegilodecakin+FOLFOX was 3(14%) with CRs in 2(9%) patients. Conclusions Pegilodecakin+FOLFOX had an acceptable tolerability profile in PDAC, with no substantial irAEs seen, and promising efficacy with the combination yielding a 2-year OS of 24% (95% CI 10-42). These data led to the phase 3 study with pegilodecakin+FOLFOX as second-line therapy of PDAC (SEQUOIA).
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Interleucina-10/uso terapéutico , Neoplasias Pancreáticas/tratamiento farmacológico , Polietilenglicoles/uso terapéutico , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/inmunología , Relación Dosis-Respuesta a Droga , Fluorouracilo/administración & dosificación , Fluorouracilo/efectos adversos , Fluorouracilo/inmunología , Fluorouracilo/uso terapéutico , Humanos , Interleucina-10/administración & dosificación , Interleucina-10/efectos adversos , Interleucina-10/inmunología , Estimación de Kaplan-Meier , Leucovorina/administración & dosificación , Leucovorina/efectos adversos , Leucovorina/inmunología , Leucovorina/uso terapéutico , Persona de Mediana Edad , Estadificación de Neoplasias , Compuestos Organoplatinos/administración & dosificación , Compuestos Organoplatinos/efectos adversos , Compuestos Organoplatinos/inmunología , Compuestos Organoplatinos/uso terapéutico , Polietilenglicoles/administración & dosificación , Polietilenglicoles/efectos adversos , Supervivencia sin Progresión , Criterios de Evaluación de Respuesta en Tumores Sólidos , Neoplasias PancreáticasRESUMEN
Purpose: Retinopathy of prematurity (ROP) is a severe complication of premature infants, leading to vision loss when untreated. Presently, the molecular mechanisms underlying ROP are still far from being clearly understood. This study sought to investigate whether thyroid hormone (TH) signaling contributes to the neuropathology of ROP using the mouse model of ROP to evaluate longitudinal photoreceptor function. Methods: Animals were exposed to hyperoxia from P7 to P12 to induce retinopathy, thereafter the animals were returned to room air (normoxia). The thyroid-activating enzyme type 2 deiodinases (Dio2) knockout (KO) mice and the littermate controls that were exposed to hyperoxia or maintained in room air and were then analyzed. The retinal function was evaluated using electroretinograms (ERGs) at three and seven weeks followed by histologic assessments with neuronal markers to detect cellular changes in the retina. Rhodopsin protein levels were measured to validate the results obtained from the immunofluorescence analyses. Results: In the ROP group, the photoreceptor ERG responses are considerably lower both in the control and the Dio2 KO animals at P23 compared to the non-ROP group. In agreement with the ERG responses, loss of Dio2 results in mislocalized cone nuclei, and abnormal rod bipolar cell dendrites extending into the outer nuclear layer. The retinal function is compromised in the adult Dio2 KO animals, although the cellular changes are less severe. Despite the reduction in scotopic a-wave amplitudes, rhodopsin levels are similar in the adult mice, across all genotypes irrespective of exposure to hyperoxia. Conclusions: Using the mouse model of ROP, we show that loss of Dio2 exacerbates the effects of hyperoxia-induced retinal deficits that persist in the adults. Our data suggest that aberrant Dio2/TH signaling is an important factor in the pathophysiology of the visual dysfunction observed in the oxygen-induced retinopathy model of ROP.
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Modelos Animales de Enfermedad , Yoduro Peroxidasa/fisiología , Células Fotorreceptoras de Vertebrados/enzimología , Retinopatía de la Prematuridad/enzimología , Glándula Tiroides/enzimología , Animales , Animales Recién Nacidos , Western Blotting , Electrorretinografía , Activadores de Enzimas , Hiperoxia/patología , Inmunohistoquímica , Ratones , Ratones Noqueados , Ratones Transgénicos , Oxígeno/metabolismo , Células Fotorreceptoras de Vertebrados/fisiología , Retinopatía de la Prematuridad/fisiopatología , Rodopsina/metabolismo , Yodotironina Deyodinasa Tipo IIRESUMEN
Particulate matter ≤2.5µm (PM2.5) air pollution is a leading environmental risk factor contributing disproportionately to the global burden of non-communicable disease. We compared impact of chronic exposure to PM2.5 alone, or with light at night exposure (LL) on metabolism. PM2.5 induced peripheral insulin resistance, circadian rhythm (CR) dysfunction, and metabolic and brown adipose tissue (BAT) dysfunction, akin to LL (with no additive interaction between PM2.5 and LL). Transcriptomic analysis of liver and BAT revealed widespread but unique alterations in CR genes, with evidence for differentially accessible promoters and enhancers of CR genes in response to PM2.5 by ATAC-seq. The histone deacetylases 2, 3, and 4 were downregulated with PM2.5 exposure, with increased promoter occupancy by the histone acetyltransferase p300 as evidenced by ChIP-seq. These findings suggest a previously unrecognized role of PM2.5 in promoting CR disruption and metabolic dysfunction through epigenetic regulation of circadian targets.
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To date, there are no effective antiviral medications for COVID-19. Drug repurposing, a strategy that uses existing drugs, offers potential prevention and treatment options for COVID-19. We discuss one treatment strategy that combines anti-inflammatory (melatonin) and antiviral (toremifene) agents for patients infected with SARS-CoV-2 from network medicine-based findings. We also describe the pathobiology and immunologic characteristics of COVID-19 and highlight the rationale of combination drug treatment to rescue the pulmonary and cardiovascular conditions resulting from COVID-19. A preliminary analysis reveals a high potential for the synergistic effects of melatonin and toremifene to reduce viral infection and replication, and the aberrant host inflammatory responses, offering strong biologic plausibility as an effective therapy for COVID-19.
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Almost all life forms can detect and decode light information for adaptive advantage. Examples include the visual system, in which photoreceptor signals are processed into virtual images, and the circadian system, in which light entrains a physiological clock. Here we describe a light response pathway in mice that employs encephalopsin (OPN3, a 480 nm, blue-light-responsive opsin) to regulate the function of adipocytes. Germline null and adipocyte-specific conditional null mice show a light- and Opn3-dependent deficit in thermogenesis and become hypothermic upon cold exposure. We show that stimulating mouse adipocytes with blue light enhances the lipolysis response and, in particular, phosphorylation of hormone-sensitive lipase. This response is Opn3 dependent. These data establish a key mechanism in which light-dependent, local regulation of the lipolysis response in white adipocytes regulates energy metabolism.
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Adipocitos Marrones/metabolismo , Adipocitos Marrones/efectos de la radiación , Adipocitos Blancos/metabolismo , Adipocitos Blancos/efectos de la radiación , Luz , Opsinas de Bastones/metabolismo , Termogénesis/efectos de la radiación , Animales , Frío , Metabolismo Energético/efectos de la radiación , Perfilación de la Expresión Génica , Lipólisis/efectos de la radiación , Ratones Endogámicos C57BL , Fenotipo , Fotones , Termogénesis/genéticaRESUMEN
During mouse postnatal eye development, the embryonic hyaloid vascular network regresses from the vitreous as an adaption for high-acuity vision. This process occurs with precisely controlled timing. Here, we show that opsin 5 (OPN5; also known as neuropsin)-dependent retinal light responses regulate vascular development in the postnatal eye. In Opn5-null mice, hyaloid vessels regress precociously. We demonstrate that 380-nm light stimulation via OPN5 and VGAT (the vesicular GABA/glycine transporter) in retinal ganglion cells enhances the activity of inner retinal DAT (also known as SLC6A3; a dopamine reuptake transporter) and thus suppresses vitreal dopamine. In turn, dopamine acts directly on hyaloid vascular endothelial cells to suppress the activity of vascular endothelial growth factor receptor 2 (VEGFR2) and promote hyaloid vessel regression. With OPN5 loss of function, the vitreous dopamine level is elevated and results in premature hyaloid regression. These investigations identify violet light as a developmental timing cue that, via an OPN5-dopamine pathway, regulates optic axis clearance in preparation for visual function.
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Dopamina/metabolismo , Ojo/irrigación sanguínea , Luz , Proteínas de la Membrana/metabolismo , Opsinas/metabolismo , Animales , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/antagonistas & inhibidores , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/química , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Endotelio Vascular/metabolismo , Ojo/enzimología , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Opsinas/genética , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/efectos de la radiación , Treonina/metabolismo , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/fisiología , Cuerpo Vítreo/metabolismoRESUMEN
A single pool of multipotent retinal progenitor cells give rise to the diverse cell types within the mammalian retina. Such cellular diversity is due to precise control of various cellular processes like cell specification, proliferation, differentiation, and maturation. Circadian clock genes can control the expression of key regulators of cell cycle progression and therefore can synchronize the cell cycle state of a heterogeneous population of cells. Here we show that the protein encoded by the circadian clock gene brain and muscle arnt-like protein-1 (Bmal1) is expressed in the embryonic retina and is required to regulate the timing of cell cycle exit. Accordingly, loss of Bmal1 during retinal neurogenesis results in increased S-phase entry and delayed cell cycle exit. Disruption in cell cycle kinetics affects the timely generation of the appropriate neuronal population thus leading to an overall decrease in the number of retinal ganglion cells, amacrine cells, and an increase in the number of the late-born type II cone bipolar cells as well as the Müller glia. Additionally, the mislocalized Müller cells are observed in the photoreceptor layer in the Bmal1 conditional mutants. These changes affect the functional integrity of the visual circuitry as we report a significant delay in visual evoked potential implicit time in the retina-specific Bmal1 null animals. Our results demonstrate that Bmal1 is required to maintain the balance between the neural and glial cells in the embryonic retina by coordinating the timing of cell cycle entry and exit. Thus, Bmal1 plays an essential role during retinal neurogenesis affecting both development and function of the mature retina.-Sawant, O. B., Jidigam, V. K., Fuller, R. D., Zucaro, O. F., Kpegba, C., Yu, M., Peachey, N. S., Rao, S. The circadian clock gene Bmal1 is required to control the timing of retinal neurogenesis and lamination of Müller glia in the mouse retina.
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Factores de Transcripción ARNTL/metabolismo , Células Ependimogliales/metabolismo , Neurogénesis , Retina/citología , Factores de Transcripción ARNTL/genética , Células Amacrinas/citología , Células Amacrinas/metabolismo , Animales , Ciclo Celular , Relojes Circadianos , Células Ependimogliales/citología , Potenciales Evocados Visuales , Ratones , Retina/embriología , Retina/metabolismo , Retina/fisiología , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismoRESUMEN
Normal development requires tight regulation of cell proliferation and cell death. Here, we have investigated these control mechanisms in the hyaloid vessels, a temporary vascular network in the mammalian eye that requires a Wnt/ß-catenin response for scheduled regression. We investigated whether the hyaloid Wnt response was linked to the oncogene Myc, and the cyclin-dependent kinase inhibitor CDKN1A (P21), both established regulators of cell cycle progression and cell death. Our analysis showed that the Wnt pathway co-receptors LRP5 and LRP6 have overlapping activities that mediate the Wnt/ß-catenin signaling in hyaloid vascular endothelial cells (VECs). We also showed that both Myc and Cdkn1a are downstream of the Wnt response and are required for hyaloid regression but for different reasons. Conditional deletion of Myc in VECs suppressed both proliferation and cell death. By contrast, conditional deletion of Cdkn1a resulted in VEC overproliferation that countered the effects of cell death on regression. When combined with analysis of MYC and CDKN1A protein levels, this analysis suggests that a Wnt/ß-catenin and MYC-CDKN1A pathway regulates scheduled hyaloid vessel regression.
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Apoptosis/fisiología , Proliferación Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Endotelio Vascular/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , beta Catenina/metabolismo , Animales , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Ojo/irrigación sanguínea , Células HEK293 , Humanos , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas c-myc/genética , Vía de Señalización Wnt/fisiologíaRESUMEN
Circadian clocks regulate various aspects of photoreceptor physiology, but their contribution to photoreceptor development and function is unclear. Cone photoreceptors are critical for color vision. Here, we define the molecular function of circadian activity within cone photoreceptors and reveal a role for the clock genes Bmal1 and Per2 in regulating cone spectral identity. ChIP analysis revealed that BMAL1 binds to the promoter region of the thyroid hormone (TH)-activating enzyme type 2 iodothyronine deiodinase (Dio2) and thus regulates the expression of Dio2. TH treatment resulted in a partial rescue of the phenotype caused by the loss of Bmal1, thus revealing a functional relationship between Bmal1 and Dio2 in establishing cone photoreceptor identity. Furthermore, Bmal1 and Dio2 are required to maintain cone photoreceptor functional integrity. Overall, our results suggest a mechanism by which circadian proteins can locally regulate the availability of TH and influence tissue development and function.
Asunto(s)
Factores de Transcripción ARNTL/genética , Relojes Circadianos/genética , Células Fotorreceptoras Retinianas Conos/metabolismo , Hormonas Tiroideas/metabolismo , Factores de Transcripción ARNTL/metabolismo , Envejecimiento , Secuencia de Aminoácidos , Animales , Relojes Circadianos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Yoduro Peroxidasa/química , Yoduro Peroxidasa/genética , Yoduro Peroxidasa/metabolismo , Ratones Noqueados , Opsinas/metabolismo , Proteínas Circadianas Period/metabolismo , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Triyodotironina/farmacología , Yodotironina Deyodinasa Tipo IIRESUMEN
PURPOSE: Familial exudative vitreoretinopathy (FEVR) is caused by mutations in the genes encoding low-density lipoprotein receptor-related protein (LRP5) or its interacting partners, namely frizzled class receptor 4 (FZD4) and norrin cystine knot growth factor (NDP). Mouse models for Lrp5, Fzd4, and Ndp have proven to be important for understanding the retinal pathophysiology underlying FEVR and systemic abnormalities related to defective Wnt signaling. Here, we report a new mouse mutant, tvrm111B, which was identified by electroretinogram (ERG) screening of mice generated in the Jackson Laboratory Translational Vision Research Models (TVRM) mutagenesis program. METHODS: ERGs were used to examine outer retinal physiology. The retinal vasculature was examined by in vivo retinal imaging, as well as by histology and immunohistochemistry. The tvrm111B locus was identified by genetic mapping of mice generated in a cross to DBA/2J, and subsequent sequencing analysis. Gene expression was examined by real-time PCR of retinal RNA. Bone mineral density (BMD) was examined by peripheral dual-energy X-ray absorptiometry. RESULTS: The tvrm111B allele is inherited as an autosomal recessive trait. Genetic mapping of the decreased ERG b-wave phenotype of tvrm111B mice localized the mutation to a region on chromosome 19 that included Lrp5. Sequencing of Lrp5 identified the insertion of a cytosine (c.4724_4725insC), which is predicted to cause a frameshift that disrupts the last three of five conserved PPPSPxS motifs in the cytoplasmic domain of LRP5, culminating in a premature termination. In addition to a reduced ERG b-wave, Lrp5tvrm111B homozygotes have low BMD and abnormal features of the retinal vasculature that have been reported previously in Lrp5 mutant mice, including persistent hyaloid vessels, leakage on fluorescein angiography, and an absence of the deep retinal capillary bed. CONCLUSIONS: The phenotype of the Lrp5tvrm111B mutant includes abnormalities of the retinal vasculature and of BMD. This model may be a useful resource to further our understanding of the biological role of LRP5 and to evaluate experimental therapies for FEVR or other conditions associated with LRP5 dysfunction.