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The COVID-19 pandemic's impact on preschool children's school readiness skills remains understudied. This research investigates Head Start preschool children's early numeracy, literacy, and executive function outcomes during a pandemic-affected school year. Study children (N = 336 assessed at fall baseline; N = 237-250 assessed in spring depending on outcome; fall baseline sample: mean age = 51 months; 46% Hispanic; 36% Black Non-Hispanic; 52% female) in a network of Head Start centers in four states (Nevada, New Jersey, Pennsylvania, and Wisconsin) experienced low in-person preschool exposure compared to national pre-pandemic norms. Children experienced fall to spring score gains during the pandemic-affected year of 0.05 SD in executive function, 0.27 SD in print knowledge, and 0.45-0.71 SD in early numeracy skills. Descriptively, for two of the three early numeracy domains measured, spring test score outcomes were stronger among children who attended more in-person preschool. We discuss implications for future research and policy.
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Here we genetically and functionally addressed potential pathways of Notch signalling in mediating vascular regeneration in mouse models. We first used transgenic adult mice with either gain- or loss-of-function Notch signalling in vascular endothelial cells and monitored perfusion in the hindlimb following ischaemia induced by femoral artery ligation. Mice deficient in Notch signalling showed defective perfusion recovery and expansion of collateral arteries. Transcriptomics analysis of arterial endothelial cells in the Notch mutants identified the guidance factor Sema3g as a candidate gene mediating reperfusion downstream of Notch. Studies in the retinal circulation showed the central role of SEMA3G downstream of Notch signalling in the orderly regulation of vascular patterning. These studies in multiple vascular beds show the primacy of Notch signalling and downstream generation of guidance peptides such as SEMA3G in promoting well-ordered vascular regeneration. KEY POINTS: Notch signalling is a critical mediator of revascularization. Yet the cellular processes activated during recovery following vascular injury are incompletely understood. Here we used genetic and cellular approaches in two different vascular beds and cultured endothelial cells to address the generalizability of mechanisms. By utilizing a highly reproducible murine model of hindlimb ischaemia in transgenic mice in which Notch signalling was inhibited at the transcriptional level, we demonstrated the centrality of Notch signalling in perfusion recovery and revascularization. RNA-sequencing of Notch mutants identified class 3 Semaphorins regulated by Notch signalling as downstream targets. Studies in retinal vessels and endothelial cells showed an essential role of guidance peptide Sema3g as a modulator of angiogenesis and orderly vascular patterning. The Notch to Sema3g signalling axis functions as a feedback mechanism to sculpt the growing vasculature in multiple beds.
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Semaforinas , Animales , Células Endoteliales/metabolismo , Miembro Posterior/irrigación sanguínea , Ratones , Neovascularización Fisiológica/fisiología , Receptor Notch1 , Receptores Notch/metabolismo , Semaforinas/genética , Semaforinas/metabolismo , Transducción de SeñalRESUMEN
Naturally occurring cell death is a fundamental developmental mechanism for regulating cell numbers and sculpting developing organs. This is particularly true in the nervous system, where large numbers of neurons and oligodendrocytes are eliminated via apoptosis during normal development. Given the profound impact of death upon these two major cell populations, it is surprising that developmental death of another major cell type-the astrocyte-has rarely been studied. It is presently unclear whether astrocytes are subject to significant developmental death, and if so, how it occurs. Here, we address these questions using mouse retinal astrocytes as our model system. We show that the total number of retinal astrocytes declines by over 3-fold during a death period spanning postnatal days 5-14. Surprisingly, these astrocytes do not die by apoptosis, the canonical mechanism underlying the vast majority of developmental cell death. Instead, we find that microglia engulf astrocytes during the death period to promote their developmental removal. Genetic ablation of microglia inhibits astrocyte death, leading to a larger astrocyte population size at the end of the death period. However, astrocyte death is not completely blocked in the absence of microglia, apparently due to the ability of astrocytes to engulf each other. Nevertheless, mice lacking microglia showed significant anatomical changes to the retinal astrocyte network, with functional consequences for the astrocyte-associated vasculature leading to retinal hemorrhage. These results establish a novel modality for naturally occurring cell death and demonstrate its importance for the formation and integrity of the retinal gliovascular network.
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Astrocitos/citología , Muerte Celular/genética , Microglía/citología , Retina/citología , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/fisiopatología , Comunicación Celular , Recuento de Células , Toxina Diftérica/toxicidad , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/efectos de los fármacos , Microglía/metabolismo , Factor de Transcripción PAX2/genética , Factor de Transcripción PAX2/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Retina/efectos de los fármacos , Retina/metabolismo , Hemorragia Retiniana/genética , Hemorragia Retiniana/metabolismo , Hemorragia Retiniana/fisiopatología , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: The risk of severe coronavirus disease 2019 (COVID-19) varies significantly among persons of similar age and is higher in males. Age-independent, sex-biased differences in susceptibility to severe COVID-19 may be ascribable to deficits in a sexually dimorphic protective attribute that we termed immunologic resilience (IR). OBJECTIVE: We sought to examine whether deficits in IR that antedate or are induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection independently predict COVID-19 mortality. METHODS: IR levels were quantified with 2 novel metrics: immune health grades (IHG-I [best] to IHG-IV) to gauge CD8+ and CD4+ T-cell count equilibrium, and blood gene expression signatures. IR metrics were examined in a prospective COVID-19 cohort (n = 522); primary outcome was 30-day mortality. Associations of IR metrics with outcomes in non-COVID-19 cohorts (n = 13,461) provided the framework for linking pre-COVID-19 IR status to IR during COVID-19, as well as to COVID-19 outcomes. RESULTS: IHG-I, tracking high-grade equilibrium between CD8+ and CD4+ T-cell counts, was the most common grade (73%) among healthy adults, particularly in females. SARS-CoV-2 infection was associated with underrepresentation of IHG-I (21%) versus overrepresentation (77%) of IHG-II or IHG-IV, especially in males versus females (P < .01). Presentation with IHG-I was associated with 88% lower mortality, after controlling for age and sex; reduced risk of hospitalization and respiratory failure; lower plasma IL-6 levels; rapid clearance of nasopharyngeal SARS-CoV-2 burden; and gene expression signatures correlating with survival that signify immunocompetence and controlled inflammation. In non-COVID-19 cohorts, IR-preserving metrics were associated with resistance to progressive influenza or HIV infection, as well as lower 9-year mortality in the Framingham Heart Study, especially in females. CONCLUSIONS: Preservation of immunocompetence with controlled inflammation during antigenic challenges is a hallmark of IR and associates with longevity and AIDS resistance. Independent of age, a male-biased proclivity to degrade IR before and/or during SARS-CoV-2 infection predisposes to severe COVID-19.
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COVID-19/inmunología , Infecciones por VIH/epidemiología , VIH-1/fisiología , Insuficiencia Respiratoria/epidemiología , SARS-CoV-2/fisiología , Factores Sexuales , Linfocitos T/inmunología , Adulto , Anciano , COVID-19/epidemiología , COVID-19/mortalidad , Estudios de Cohortes , Resistencia a la Enfermedad , Femenino , Humanos , Inmunocompetencia , Interleucina-6/sangre , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Índice de Severidad de la Enfermedad , Análisis de Supervivencia , Transcriptoma/inmunología , Estados Unidos/epidemiología , Carga ViralRESUMEN
BACKGROUND: Cardiac rehabilitation programs (CRP) are effective evidence-based secondary prevention programs that reduce morbidity and mortality in patients with cardiovascular disease (CVD). However, participation remains suboptimal, resulting in under-treatment and greater risk for recurrent cardiac events. Understanding the reasons behind CRP dropout is urgently needed to inform the development of programs that best meet patient needs and support sustained engagement. AIMS: The aim of this study was to identify and understand factors impacting CRP dropout from the patient perspective. METHODS: A qualitative study using semi-structured interviews was undertaken to examine the experience of 23 patients who dropped out of a CRP within a large urban hospital in British Columbia, Canada. Data were coded, analyzed using the constant comparison technique, and organized thematically. RESULTS: Participants described multiple challenges when attempting to complete CRP. Analysis of the data led to the identification of three main categories: (1) challenges living with CVD, (2) perceived advantages and disadvantages of CRP, and (3) unmet needs during CRP. LINKING EVIDENCE TO ACTION: In the practice setting, assessment of readiness to engage in CRP, alongside patient preferences and engagement needs, should be undertaken for maximum CRP uptake and completion. Providing diverse modes of CRP delivery, along with exploring the impact of virtual options as compared to traditional in-person programs, will further advance the CRP evidence and may help address pervasive access barriers.
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Rehabilitación Cardiaca , Enfermedades Cardiovasculares , Colombia Británica , Rehabilitación Cardiaca/métodos , Humanos , Evaluación del Resultado de la Atención al Paciente , Investigación CualitativaRESUMEN
PURPOSE: In order to minimize the risk of transmission of coronavirus disease 2019 (COVID-19), many clinic appointments were postponed to lower patient attendance. Actively calling patients to postpone appointments is a labour-intensive process. We were the first ophthalmic clinics in Hong Kong to use short message service (SMS) to dispatch messages simultaneously to a large number of patients to offer postponement of appointments. The aim of this study is to evaluate whether SMS is an effective method to reduce outpatient attendance during the COVID-19 pandemic. METHODS: This is an observational study reviewing data on SMS messages sent to all patients attending ophthalmology clinics of a tertiary eye centre in Hong Kong. All SMS were sent at least 5 days before the scheduled appointments. The text message included an enquiry hotline for postponement of appointments and offered drug refill. The study included data from February to April 2020. Two hundred patients were invited to take part in a questionnaire on satisfaction level and reason(s) for appointment rescheduling. RESULTS: During the study period, a total of 17,028 SMS were sent. The overall response rate was 23.6%. 14.3% postponed their appointments. This led to an overall 13.9% reduction of clinic attendance. The overall satisfaction was high (96%). The main reason for postponing appointment was worries about infection risk (93.1%). CONCLUSION: SMS was an efficient and cost-effective flow-control method which was well accepted by patients and can reduce outpatient attendance. The time saved can potentially allow healthcare workers to conduct other infection control measures during the COVID-19 pandemic.
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COVID-19/epidemiología , Oftalmopatías/epidemiología , Pandemias , Cooperación del Paciente/estadística & datos numéricos , Envío de Mensajes de Texto , Comorbilidad , Femenino , Hong Kong/epidemiología , Humanos , Masculino , SARS-CoV-2RESUMEN
Lipid droplets (LD) are dynamic organelles involved in intracellular lipid metabolism in almost all eukaryotic cells, and LD-associated proteins tightly regulate their dynamics. One LD coat protein is caveolin-1 (Cav-1), an essential component for caveola assembly in highly differentiated cells, including adipocytes, smooth muscle cells, and endothelial cells (EC). However, the role of Cav-1 in LD dynamics is unclear. Here we report that EC lacking Cav-1 exhibit impaired LD formation. The decreased LD formation is due to enhanced lipolysis and not caused by reduced triglyceride synthesis or fatty acid uptake. Mechanistically, the absence of Cav-1 increased cAMP/PKA signaling in EC, as indicated by elevated phosphorylation of hormone-sensitive lipase and increased lipolysis. Unexpectedly, we also observed enhanced autocrine production of prostaglandin I2 (PGI2, also called prostacyclin) in Cav-1 KO EC, and this PGI2 increase appeared to stimulate cAMP/PKA pathways, contributing to the enhanced lipolysis in Cav-1 KO cells. Our results reveal an unanticipated role of Cav-1 in regulating lipolysis in non-adipose tissue, indicating that Cav-1 is required for LD metabolism in EC and that it regulates cAMP-dependent lipolysis in part via the autocrine production of PGI2.
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Caveolina 1/metabolismo , AMP Cíclico/metabolismo , Células Endoteliales/metabolismo , Epoprostenol/farmacología , Lipólisis/efectos de los fármacos , Animales , Western Blotting , Caveolina 1/genética , Línea Celular , Células Cultivadas , Metabolismo de los Lípidos/efectos de los fármacos , Espectrometría de Masas , Ratones , Ratones Mutantes , FosforilaciónRESUMEN
RATIONALE: Fatty acids (FA) are transported across the capillary endothelium to parenchymal tissues. However, it is not known how endothelial cells (EC) from large vessels process a postprandial surge of FA. OBJECTIVE: This study was designed to characterize lipid droplet (LD) formation in EC by manipulating pathways leading to the formation and degradation of LD. In addition, several functions of LD-derived FA were assessed. METHODS AND RESULTS: LD were present in EC lining the aorta after the peak in plasma triglycerides initiated by a gavage of olive oil in mice, in vivo. Similarly, in isolated aorta, oleic acid treatment generates LD in EC ex vivo. Cultured EC readily form LD largely via the enzyme DGAT (diacylglycerol O-acyltransferase 1) and degrade LD via ATGL (adipocyte triglyceride lipase) after FA loading. Functionally, LD-derived FA are dynamically regulated and function to protect EC from lipotoxic stress and provide FA for metabolic needs. CONCLUSIONS: Our results delineate endothelial LD dynamics for the first time in vivo and in vitro. Moreover, LD formation protects EC from lipotoxic stress, regulates EC glycolysis, and provides a source of FA for adjacent cells in the vessel wall or tissues.
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Aorta Torácica/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Gotas Lipídicas/metabolismo , Aceite de Oliva/metabolismo , Administración Oral , Animales , Células Cultivadas , Diacilglicerol O-Acetiltransferasa/metabolismo , Glucólisis , Humanos , Hidrólisis , Intubación Gastrointestinal , Lipasa/metabolismo , Lipólisis , Ratones Endogámicos C57BL , Ácido Oléico/metabolismo , Aceite de Oliva/administración & dosificación , Factores de Tiempo , Triglicéridos/sangreRESUMEN
OBJECTIVE: The importance of PI3K/Akt signaling in the vasculature has been demonstrated in several models, as global loss of Akt1 results in impaired postnatal ischemia- and VEGF-induced angiogenesis. The ubiquitous expression of Akt1, however, raises the possibility of cell-type-dependent Akt1-driven actions, thereby necessitating tissue-specific characterization. APPROACH AND RESULTS: Herein, we used an inducible, endothelial-specific Akt1-deleted adult mouse model (Akt1iECKO) to characterize the endothelial cell autonomous functions of Akt1 in the vascular system. Endothelial-targeted ablation of Akt1 reduces eNOS (endothelial nitric oxide synthase) phosphorylation and promotes both increased vascular contractility in isolated vessels and elevated diastolic blood pressures throughout the diurnal cycle in vivo. Furthermore, Akt1iECKO mice subject to the hindlimb ischemia model display impaired blood flow and decreased arteriogenesis. CONCLUSIONS: Endothelial Akt1 signaling is necessary for ischemic resolution post-injury and likely reflects the consequence of NO insufficiency critical for vascular repair.
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Aorta Torácica/enzimología , Células Endoteliales/enzimología , Isquemia/enzimología , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Vasoconstricción , Animales , Velocidad del Flujo Sanguíneo , Presión Sanguínea , Modelos Animales de Enfermedad , Miembro Posterior , Isquemia/genética , Isquemia/patología , Isquemia/fisiopatología , Masculino , Ratones Noqueados , Músculo Esquelético/enzimología , Músculo Esquelético/patología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/deficiencia , Proteínas Proto-Oncogénicas c-akt/genética , Flujo Sanguíneo Regional , Transducción de SeñalRESUMEN
CONTEXT: As the utility of point-of-care ultrasound (POCUS) continues to expand in the medical field, there is a need for effective educational methods. In Switzerland, medical education follows the European model and lasts 6 years, focusing on preclinical training during the first 2 years. No previous studies have evaluated the optimal time for teaching ultrasound in European medical education. AIMS: The aim of this study is to provide ultrasound training to medical students in Switzerland at varying times during their clinical training to determine if the level of training plays a role in their ability to comprehend and to apply basic POCUS skills. METHODS: We performed an observational study utilizing a convenience sample of Swiss medical students between July 11, 2016 and August 6, 2016. They were taught a 2-day POCUS course by five American-trained 1st-year medical students. Following this course, students were evaluated with written and clinical examination. RESULTS: 100 Swiss medical students were enrolled in the study. A total of 59 of these students were early clinical students, and 41 students were late clinical students. A two-tailed t-test was performed and demonstrated that the late clinical students performed better than the early clinical students on the written assessment; however, no difference was found in clinical skill. CONCLUSION: Our data suggest that Swiss medical students can learn and perform POCUS after a 2-day instructional taught by trained 1st-year American medical students. No difference was found between students in early clinical training and late clinical training for the ability to perform POCUS.
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RATIONALE: Caveolin-1 (Cav-1) negatively regulates endothelial nitric oxide (NO) synthase-derived NO production, and this has been mapped to several residues on Cav-1, including F92. Herein, we reasoned that endothelial expression of an F92ACav-1 transgene would let us decipher the mechanisms and relationships between caveolae structure and intracellular signaling. OBJECTIVE: This study was designed to separate caveolae formation from its downstream signaling effects. METHODS AND RESULTS: An endothelial-specific doxycycline-regulated mouse model for the expression of Cav-1-F92A was developed. Blood pressure by telemetry and nitric oxide bioavailability by electron paramagnetic resonance and phosphorylation of vasodilator-stimulated phosphoprotein were determined. Caveolae integrity in the presence of Cav-1-F92A was measured by stabilization of caveolin-2, sucrose gradient, and electron microscopy. Histological analysis of heart and lung, echocardiography, and signaling were performed. CONCLUSIONS: This study shows that mutant Cav-1-F92A forms caveolae structures similar to WT but leads to increases in NO bioavailability in vivo, thereby demonstrating that caveolae formation and downstream signaling events occur through independent mechanisms.
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Caveolas/metabolismo , Caveolina 1/biosíntesis , Caveolina 1/genética , Líquido Intracelular/metabolismo , Transducción de Señal/fisiología , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Caveolas/efectos de los fármacos , Doxiciclina/farmacología , Humanos , Líquido Intracelular/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Óxido Nítrico/metabolismo , Transducción de Señal/efectos de los fármacos , Desacopladores/farmacologíaRESUMEN
RATIONALE: Several lines of evidence indicate that the regulation of microRNA (miRNA) levels by different stimuli may contribute to the modulation of stimulus-induced responses. The miR-17-92 cluster has been linked to tumor development and angiogenesis, but its role in vascular endothelial growth factor-induced endothelial cell (EC) functions is unclear and its regulation is unknown. OBJECTIVE: The purpose of this study was to elucidate the mechanism by which VEGF regulates the expression of miR-17-92 cluster in ECs and determine its contribution to the regulation of endothelial angiogenic functions, both in vitro and in vivo. This was done by analyzing the effect of postnatal inactivation of miR-17-92 cluster in the endothelium (miR-17-92 iEC-KO mice) on developmental retinal angiogenesis, VEGF-induced ear angiogenesis, and tumor angiogenesis. METHODS AND RESULTS: Here, we show that Erk/Elk1 activation on VEGF stimulation of ECs is responsible for Elk-1-mediated transcription activation (chromatin immunoprecipitation analysis) of the miR-17-92 cluster. Furthermore, we demonstrate that VEGF-mediated upregulation of the miR-17-92 cluster in vitro is necessary for EC proliferation and angiogenic sprouting. Finally, we provide genetic evidence that miR-17-92 iEC-KO mice have blunted physiological retinal angiogenesis during development and diminished VEGF-induced ear angiogenesis and tumor angiogenesis. Computational analysis and rescue experiments show that PTEN (phosphatase and tensin homolog) is a target of the miR-17-92 cluster and is a crucial mediator of miR-17-92-induced EC proliferation. However, the angiogenic transcriptional program is reduced when miR-17-92 is inhibited. CONCLUSIONS: Taken together, our results indicate that VEGF-induced miR-17-92 cluster expression contributes to the angiogenic switch of ECs and participates in the regulation of angiogenesis.
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Endotelio Vascular/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , MicroARNs/biosíntesis , Neovascularización Fisiológica/fisiología , Factor A de Crecimiento Endotelial Vascular/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Endotelio Vascular/efectos de los fármacos , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Noqueados , MicroARNs/genética , Neovascularización Fisiológica/efectos de los fármacosRESUMEN
OBJECTIVE: Drug-eluting stent delivery of mTORC1 (mechanistic target of rapamycin complex 1) inhibitors is highly effective in preventing intimal hyperplasia after coronary revascularization, but adverse effects limit their use for systemic vascular disease. Understanding the mechanism of action may lead to new treatment strategies. We have shown that rapamycin promotes vascular smooth muscle cell differentiation in an AKT2-dependent manner in vitro. Here, we investigate the roles of AKT (protein kinase B) isoforms in intimal hyperplasia. APPROACH AND RESULTS: We found that germ-line-specific or smooth muscle-specific deletion of Akt2 resulted in more severe intimal hyperplasia compared with control mice after arterial denudation injury. Conversely, smooth muscle-specific Akt1 knockout prevented intimal hyperplasia, whereas germ-line Akt1 deletion caused severe thrombosis. Notably, rapamycin prevented intimal hyperplasia in wild-type mice but had no therapeutic benefit in Akt2 knockouts. We identified opposing roles for AKT1 and AKT2 isoforms in smooth muscle cell proliferation, migration, differentiation, and rapamycin response in vitro. Mechanistically, rapamycin induced MYOCD (myocardin) mRNA expression. This was mediated by AKT2 phosphorylation and nuclear exclusion of FOXO4 (forkhead box O4), inhibiting its binding to the MYOCD promoter. CONCLUSIONS: Our data reveal opposing roles for AKT isoforms in smooth muscle cell remodeling. AKT2 is required for rapamycin's therapeutic inhibition of intimal hyperplasia, likely mediated in part through AKT2-specific regulation of MYOCD via FOXO4. Because AKT2 signaling is impaired in diabetes mellitus, this work has important implications for rapamycin therapy, particularly in diabetic patients.
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Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Neointima , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sirolimus/farmacología , Lesiones del Sistema Vascular/prevención & control , Animales , Sitios de Unión , Proteínas de Ciclo Celular , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Ratones Noqueados , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/lesiones , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/enzimología , Miocitos del Músculo Liso/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenotipo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-akt/deficiencia , Proteínas Proto-Oncogénicas c-akt/genética , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Lesiones del Sistema Vascular/enzimología , Lesiones del Sistema Vascular/genética , Lesiones del Sistema Vascular/patologíaRESUMEN
Models of unregulated nitric oxide (NO) diffusion do not consistently account for the biochemistry of NO synthase (NOS)-dependent signalling in many cell systems. For example, endothelial NOS controls blood pressure, blood flow and oxygen delivery through its effect on vascular smooth muscle tone, but the regulation of these processes is not adequately explained by simple NO diffusion from endothelium to smooth muscle. Here we report a new model for the regulation of NO signalling by demonstrating that haemoglobin (Hb) α (encoded by the HBA1 and HBA2 genes in humans) is expressed in human and mouse arterial endothelial cells and enriched at the myoendothelial junction, where it regulates the effects of NO on vascular reactivity. Notably, this function is unique to Hb α and is abrogated by its genetic depletion. Mechanistically, endothelial Hb α haem iron in the Fe(3+) state permits NO signalling, and this signalling is shut off when Hb α is reduced to the Fe(2+) state by endothelial cytochrome b5 reductase 3 (CYB5R3, also known as diaphorase 1). Genetic and pharmacological inhibition of CYB5R3 increases NO bioactivity in small arteries. These data reveal a new mechanism by which the regulation of the intracellular Hb α oxidation state controls NOS signalling in non-erythroid cells. This model may be relevant to haem-containing globins in a broad range of NOS-containing somatic cells.
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Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Hemoglobinas/metabolismo , Óxido Nítrico/metabolismo , Fragmentos de Péptidos/metabolismo , Transducción de Señal , Agonistas de Receptores Adrenérgicos alfa 1/farmacología , Animales , Células Cultivadas , Difusión , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Hemoglobinas/genética , Humanos , Hierro/química , Ratones , Óxido Nítrico Sintasa/metabolismo , Oxidación-Reducción , Fragmentos de Péptidos/genética , Fenilefrina/farmacologíaRESUMEN
The contribution of endothelial-derived miR-17â¼92 to ischemia-induced arteriogenesis has not been investigated in an in vivo model. In the present study, we demonstrate a critical role for the endothelial-derived miR-17â¼92 cluster in shaping physiological and ischemia-triggered arteriogenesis. Endothelial-specific deletion of miR-17â¼92 results in an increase in collateral density limbs and hearts and in ischemic limbs compared with control mice, and consequently improves blood flow recovery. Individual cluster components positively or negatively regulate endothelial cell (EC) functions in vitro, and, remarkably, ECs lacking the cluster spontaneously form cords in a manner rescued by miR-17a, -18a, and -19a. Using both in vitro and in vivo analyses, we identified FZD4 and LRP6 as targets of miR-19a/b. Both of these targets were up-regulated in 17â¼92 KO ECs compared with control ECs, and both were shown to be targeted by miR-19 using luciferase assays. We demonstrate that miR-19a negatively regulates FZD4, its coreceptor LRP6, and WNT signaling, and that antagonism of miR-19a/b in aged mice improves blood flow recovery after ischemia and reduces repression of these targets. Collectively, these data provide insights into miRNA regulation of arterialization and highlight the importance of vascular WNT signaling in maintaining arterial blood flow.
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Receptores Frizzled/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , MicroARNs/metabolismo , Familia de Multigenes/fisiología , Neovascularización Fisiológica/fisiología , Vía de Señalización Wnt/fisiología , Animales , Receptores Frizzled/genética , Isquemia/genética , Isquemia/metabolismo , Isquemia/patología , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Ratones , Ratones Noqueados , MicroARNs/genéticaRESUMEN
INTRODUCTION: A consensus statement proposed a diagnostic framework to systematise the identification of paroxysmal sympathetic hyperactivity (PSH) using the PSH-Assessment Measure (PSH-AM). METHODS: This retrospective study identified adult patients with a primary diagnosis of traumatic brain injury and a hospital length of stay >14 days. Based on PSH-AM scores, patients were grouped into 'unlikely', 'possible', or 'probable' PSH. For this study, 'possible' and 'probable' PSH patients were collapsed into a single group (PSH+), and resultant data were compared with 'unlikely' diagnoses (PSH-). PSH-AM data were assessed against clinical diagnoses to establish sensitivity and specificity data. RESULTS: Sixty five patients met inclusion criteria, with 45/65 (69%) categorised as either 'possible' or 'probable' PSH on the PSH-AM. Only 16 of these patients were diagnosed by clinicians. The most common symptoms triggering clinical diagnosis were tachycardia, fever and posturing. Increased respiratory rate, blood pressure or the presence of diaphoresis were not used in diagnosing PSH if the PSH-AM was not utilised. Assuming clinical assessment as the current gold standard, the PSH-AM yielded a sensitivity of 94% and a specificity of 35% when used retrospectively. Patients clinically diagnosed with PSH were discharged 5 days earlier compared to those identified by the PSH-AM. CONCLUSIONS: The recently proposed diagnostic framework may reduce misdiagnosis, length of stay and hospitalisation costs.
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Enfermedades del Sistema Nervioso Autónomo/diagnóstico , Enfermedades del Sistema Nervioso Autónomo/etiología , Lesiones Traumáticas del Encéfalo/complicaciones , Evaluación de Resultado en la Atención de Salud , Adolescente , Adulto , Enfermedades del Sistema Nervioso Autónomo/tratamiento farmacológico , Enfermedades del Sistema Nervioso Autónomo/epidemiología , Lesiones Traumáticas del Encéfalo/epidemiología , Estudios de Cohortes , Femenino , Escala de Coma de Glasgow , Humanos , Incidencia , Tiempo de Internación , Masculino , Persona de Mediana Edad , Estadísticas no Paramétricas , Tomógrafos Computarizados por Rayos X , Adulto JovenRESUMEN
Here we show that dynamin 2 (Dnm2) is essential for angiogenesis in vitro and in vivo. In cultured endothelial cells lacking Dnm2, vascular endothelial growth factor (VEGF) signaling and receptor levels are augmented whereas cell migration and morphogenesis are impaired. Mechanistically, the loss of Dnm2 increases focal adhesion size and the surface levels of multiple integrins and reduces the activation state of ß1 integrin. In vivo, the constitutive or inducible loss of Dnm2 in endothelium impairs branching morphogenesis and promotes the accumulation of ß1 integrin at sites of failed angiogenic sprouting. Collectively, our data show that Dnm2 uncouples VEGF signaling from function and coordinates the endocytic turnover of integrins in a manner that is crucially important for angiogenesis in vitro and in vivo.
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Vasos Sanguíneos/embriología , Dinamina II/fisiología , Endocitosis/genética , Integrinas/metabolismo , Neovascularización Fisiológica/genética , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Animales Recién Nacidos , Vasos Sanguíneos/crecimiento & desarrollo , Células Cultivadas , Dinamina II/genética , Embrión de Mamíferos , Femenino , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Masculino , Ratones , Ratones Transgénicos , Transducción de Señal/fisiologíaRESUMEN
The PI3K/Akt pathway is necessary for several key endothelial cell (EC) functions, including cell growth, migration, survival, and vascular tone. However, existing literature supports the idea that Akt can be either pro- or antiangiogenic, possibly due to compensation by multiple isoforms in the EC when a single isoform is deleted. Thus, biochemical, genetic, and proteomic studies were conducted to examine isoform-substrate specificity for Akt1 vs. Akt2. In vitro, Akt1 preferentially phosphorylates endothelial nitric oxide synthase (eNOS) and promotes NO release, whereas nonphysiological overexpression of Akt2 can bypass the loss of Akt1. Conditional deletion of Akt1 in the EC, in the absence or presence of Akt2, retards retinal angiogenesis, implying that Akt1 exerts a nonredundant function during physiological angiogenesis. Finally, proteomic analysis of Akt substrates isolated from Akt1- or Akt2-deficient ECs documents that phosphorylation of multiple Akt substrates regulating angiogenic signaling is reduced in Akt1-deficient, but not Akt2-deficient, ECs, including eNOS and Forkhead box proteins. Therefore, Akt1 promotes angiogenesis largely due to phosphorylation and regulation of important downstream effectors that promote aspects of angiogenic signaling.
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Endotelio Vascular/metabolismo , Neovascularización Fisiológica/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Vasos Retinianos/metabolismo , Animales , Línea Celular Transformada , Pulmón/irrigación sanguínea , Pulmón/citología , Ratones , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo III/metabolismo , Compuestos de Mostaza Nitrogenada/metabolismo , Fosforilación/fisiología , Proteómica , Proteínas Proto-Oncogénicas c-akt/genética , Retina/patología , Vasos Retinianos/patología , Transducción de Señal/fisiología , Especificidad por SustratoRESUMEN
Vascular remodeling is essential for tissue repair and is regulated by multiple factors, including thrombospondin-2 (TSP2) and hypoxia/VEGF-induced activation of Akt. In contrast to TSP2 knock-out (KO) mice, Akt1 KO mice have elevated TSP2 expression and delayed tissue repair. To investigate the contribution of increased TSP2 to Akt1 KO mice phenotypes, we generated Akt1/TSP2 double KO (DKO) mice. Full-thickness excisional wounds in DKO mice healed at an accelerated rate when compared with Akt1 KO mice. Isolated dermal Akt1 KO fibroblasts expressed increased TSP2 and displayed altered morphology and defects in migration and adhesion. These defects were rescued in DKO fibroblasts or after TSP2 knockdown. Conversely, the addition of exogenous TSP2 to WT cells induced cell morphology and migration rates that were similar to those of Akt1 KO cells. Akt1 KO fibroblasts displayed reduced adhesion to fibronectin with manganese stimulation when compared with WT and DKO cells, revealing an Akt1-dependent role for TSP2 in regulating integrin-mediated adhesions; however, this effect was not due to changes in ß1 integrin surface expression or activation. Consistent with these results, Akt1 KO fibroblasts displayed reduced Rac1 activation that was dependent upon expression of TSP2 and could be rescued by a constitutively active Rac mutant. Our observations show that repression of TSP2 expression is a critical aspect of Akt1 function in tissue repair.
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Fibroblastos/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Piel/metabolismo , Trombospondinas/genética , Heridas no Penetrantes/genética , Animales , Movimiento Celular , Fibroblastos/patología , Regulación de la Expresión Génica , Prueba de Complementación Genética , Integrina beta1/genética , Integrina beta1/metabolismo , Ratones , Ratones Noqueados , Neuropéptidos/genética , Neuropéptidos/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/deficiencia , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Piel/lesiones , Piel/patología , Trombospondinas/deficiencia , Cicatrización de Heridas/genética , Heridas no Penetrantes/metabolismo , Heridas no Penetrantes/patología , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Cerebral microvascular disease predominantly affects brain white matter and deep grey matter, resulting in ischaemic damage that ranges from lacunar infarcts to white matter hyperintensities seen on magnetic resonance imaging. These lesions are common and result in both clinical stroke syndromes and accumulate over time, resulting in cognitive deficits and dementia. Magnetic resonance imaging studies suggest that these lesions progress over time, accumulate adjacent to prior lesions and have a penumbral region susceptible to further injury. The pathological correlates of this adjacent injury in surviving myelinated axons have not been previously defined. In this study, we sought to determine the molecular organization of axons in tissue adjacent to lacunar infarcts and in the regions surrounding microinfarcts, by determining critical elements in axonal function: the morphology and length of node of Ranvier segments and adjacent paranodal segments. We examined post-mortem brain tissue from six patients with lacunar infarcts and tissue from two patients with autosomal dominant retinal vasculopathy and cerebral leukoencephalopathy (previously known as hereditary endotheliopathy with retinopathy, nephropathy and stroke) who accumulate progressive white matter ischaemic lesions in the form of lacunar and microinfarcts. In axons adjacent to lacunar infarcts yet extending up to 150% of the infarct diameter away, both nodal and paranodal length increase by â¼20% and 80%, respectively, reflecting a loss of normal cell-cell adhesion and signalling between axons and oligodendrocytes. Using premorbid magnetic resonance images, brain regions from patients with retinal vasculopathy and cerebral leukoencephalopathy that harboured periventricular white matter hyperintensities were selected and the molecular organization of axons was determined within these regions. As in regions adjacent to lacunar infarcts, nodal and paranodal length in white matter of these patients is increased. Myelin basic protein and neurofilament immunolabelling demonstrates that axons in these adjacent regions have preserved axonal cytoskeleton organization and are generally myelinated. This indicates that the loss of normal axonal microdomain architecture results from disrupted axoglial signalling in white matter adjacent to lacunar and microinfarcts. The loss of the normal molecular organization of nodes and paranodes is associated with axonal degeneration and may lead to impaired conduction velocity across surviving axons after stroke. These findings demonstrate that the degree of white matter injury associated with cerebral microvascular disease extends well beyond what can be identified using imaging techniques and that an improved understanding of the neurobiology in these regions can drive new therapeutic strategies for this disease entity.