RESUMEN
The innate immune response is essential for combating infectious disease. Macrophages and other cells respond to infection by releasing cytokines, such as interleukin-1ß (IL-1ß), which in turn activate a well-described, myeloid-differentiation factor 88 (MYD88)-mediated, nuclear factor-κB (NF-κB)-dependent transcriptional pathway that results in inflammatory-cell activation and recruitment. Endothelial cells, which usually serve as a barrier to the movement of inflammatory cells out of the blood and into tissue, are also critical mediators of the inflammatory response. Paradoxically, the cytokines vital to a successful immune defence also have disruptive effects on endothelial cell-cell interactions and can trigger degradation of barrier function and dissociation of tissue architecture. The mechanism of this barrier dissolution and its relationship to the canonical NF-κB pathway remain poorly defined. Here we show that the direct, immediate and disruptive effects of IL-1ß on endothelial stability in a human in vitro cell model are NF-κB independent and are instead the result of signalling through the small GTPase ADP-ribosylation factor 6 (ARF6) and its activator ARF nucleotide binding site opener (ARNO; also known as CYTH2). Moreover, we show that ARNO binds directly to the adaptor protein MYD88, and thus propose MYD88-ARNO-ARF6 as a proximal IL-1ß signalling pathway distinct from that mediated by NF-κB. Finally, we show that SecinH3, an inhibitor of ARF guanine nucleotide-exchange factors such as ARNO, enhances vascular stability and significantly improves outcomes in animal models of inflammatory arthritis and acute inflammation.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Receptores de Interleucina/metabolismo , Factor 6 de Ribosilación del ADP , Adyuvantes Inmunológicos/farmacología , Animales , Artritis/patología , Cadherinas/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Línea Celular , Células Endoteliales/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Interleucina-1beta/farmacología , FN-kappa B/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Transporte de Proteínas/efectos de los fármacos , Purinas/farmacología , Transducción de Señal , Tiofenos/farmacologíaRESUMEN
Hoxa3 was the first Hox gene to be mutated by gene targeting in mice and is required for the development of multiple endoderm and neural crest cell (NCC)-derived structures in the pharyngeal region. Previous studies have shown that the Hoxa3 null mutant lacks third pharyngeal pouch derivatives, the thymus and parathyroids by E18.5, and organ-specific markers are absent or downregulated during initial organogenesis. Our current analysis of the Hoxa3 null mutant shows that organ-specific domains did undergo initial patterning, but the location and timing of key regional markers within the pouch, including Tbx1, Bmp4 and Fgf8, were altered. Expression of the parathyroid marker Gcm2 was initiated but was quickly downregulated and differentiation failed; by contrast, thymus markers were delayed but achieved normal levels, concurrent with complete loss through apoptosis. To determine the cell type-specific roles of Hoxa3 in third pharyngeal pouch development, we analyzed tissue-specific mutants using endoderm and/or NCC-specific Cre drivers. Simultaneous deletion with both drivers resulted in athymia at E18.5, similar to the null. By contrast, the individual tissue-specific Hoxa3 deletions resulted in small, ectopic thymi, although each had a unique phenotype. Hoxa3 was primarily required in NCCs for morphogenesis. In endoderm, Hoxa3 temporally regulated initiation of the thymus program and was required in a cell-autonomous manner for parathyroid differentiation. Furthermore, Hoxa3 was required for survival of third pharyngeal pouch-derived organs, but expression in either tissue was sufficient for this function. These data show that Hoxa3 has multiple complex and tissue-specific functions during patterning, differentiation and morphogenesis of the thymus and parathyroids.
Asunto(s)
Biomarcadores/metabolismo , Diferenciación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/metabolismo , Morfogénesis/fisiología , Glándulas Paratiroides/embriología , Timo/embriología , Animales , Apoptosis/fisiología , Región Branquial/metabolismo , Cartilla de ADN/genética , Galactósidos , Regulación del Desarrollo de la Expresión Génica/genética , Genotipo , Técnicas Histológicas , Inmunohistoquímica , Hibridación in Situ , Indoles , Ratones , Modelos Anatómicos , Morfogénesis/genética , Estadísticas no ParamétricasRESUMEN
BACKGROUND: Cerebral cavernous malformation (CCM) is a hemorrhagic stroke disease affecting up to 0.5% of North Americans that has no approved nonsurgical treatment. A subset of patients have a hereditary form of the disease due primarily to loss-of-function mutations in KRIT1, CCM2, or PDCD10. We sought to identify known drugs that could be repurposed to treat CCM. METHODS AND RESULTS: We developed an unbiased screening platform based on both cellular and animal models of loss of function of CCM2. Our discovery strategy consisted of 4 steps: an automated immunofluorescence and machine-learning-based primary screen of structural phenotypes in human endothelial cells deficient in CCM2, a secondary screen of functional changes in endothelial stability in these same cells, a rapid in vivo tertiary screen of dermal microvascular leak in mice lacking endothelial Ccm2, and finally a quaternary screen of CCM lesion burden in these same mice. We screened 2100 known drugs and bioactive compounds and identified 2 candidates, cholecalciferol (vitamin D3) and tempol (a scavenger of superoxide), for further study. Each drug decreased lesion burden in a mouse model of CCM vascular disease by ≈50%. CONCLUSIONS: By identifying known drugs as potential therapeutics for CCM, we have decreased the time, cost, and risk of bringing treatments to patients. Each drug also prompts additional exploration of biomarkers of CCM disease. We further suggest that the structure-function screening platform presented here may be adapted and scaled to facilitate drug discovery for diverse loss-of-function genetic vascular disease.
Asunto(s)
Neoplasias del Sistema Nervioso Central/tratamiento farmacológico , Modelos Animales de Enfermedad , Reposicionamiento de Medicamentos/métodos , Hemangioma Cavernoso del Sistema Nervioso Central/tratamiento farmacológico , Animales , Células Cultivadas , Neoplasias del Sistema Nervioso Central/patología , Colecalciferol/farmacología , Colecalciferol/uso terapéutico , Ensayos de Selección de Medicamentos Antitumorales/métodos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Depuradores de Radicales Libres/farmacología , Depuradores de Radicales Libres/uso terapéutico , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Resultado del TratamientoRESUMEN
TBX3 is critical for human development: mutations in TBX3 cause congenital anomalies in patients with ulnar-mammary syndrome. Data from mice and humans suggest multiple roles for Tbx3 in development and function of the cardiac conduction system. The mechanisms underlying the functional development, maturation, and maintenance of the conduction system are not well understood. We tested the requirements for Tbx3 in these processes. We generated a unique series of Tbx3 hypomorphic and conditional mouse mutants with varying levels and locations of Tbx3 activity within the heart, and developed techniques for evaluating in vivo embryonic conduction system function. Disruption of Tbx3 function in different regions of the developing heart causes discrete phenotypes and lethal arrhythmias: sinus pauses and bradycardia indicate sinoatrial node dysfunction, whereas preexcitation and atrioventricular block reveal abnormalities in the atrioventricular junction. Surviving Tbx3 mutants are at increased risk for sudden death. Arrhythmias induced by knockdown of Tbx3 in adults reveal its requirement for conduction system homeostasis. Arrhythmias in Tbx3-deficient embryos are accompanied by disrupted expression of multiple ion channels despite preserved expression of previously described conduction system markers. These findings indicate that Tbx3 is required for the conduction system to establish and maintain its correct molecular identity and functional properties. In conclusion, Tbx3 is required for the functional development, maturation, and homeostasis of the conduction system in a highly dosage-sensitive manner. TBX3 and its regulatory targets merit investigation as candidates for human arrhythmias.
Asunto(s)
Arritmias Cardíacas/fisiopatología , Dosificación de Gen , Sistema de Conducción Cardíaco/fisiopatología , Homeostasis/genética , Proteínas de Dominio T Box/deficiencia , Proteínas de Dominio T Box/genética , Alelos , Animales , Animales Recién Nacidos , Arritmias Cardíacas/complicaciones , Arritmias Cardíacas/diagnóstico por imagen , Arritmias Cardíacas/patología , Bloqueo Atrioventricular/complicaciones , Bloqueo Atrioventricular/diagnóstico por imagen , Bloqueo Atrioventricular/patología , Bloqueo Atrioventricular/fisiopatología , Nodo Atrioventricular/patología , Nodo Atrioventricular/fisiopatología , Conexina 43/metabolismo , Electrocardiografía , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/patología , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Sistema de Conducción Cardíaco/anomalías , Sistema de Conducción Cardíaco/diagnóstico por imagen , Sistema de Conducción Cardíaco/patología , Humanos , Canales Iónicos/genética , Canales Iónicos/metabolismo , Ratones , Mutación/genética , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Recombinación Genética/genética , Análisis de Supervivencia , Proteínas de Dominio T Box/metabolismo , UltrasonografíaRESUMEN
Mitoferrin1 is 1 of 2 homologous mitochondrial iron transporters and is required for mitochondrial iron delivery in developing erythroid cells. We show that total deletion of Mfrn1 in embryos leads to embryonic lethality. Selective deletion of Mfrn1 in adult hematopoietic tissues leads to severe anemia because of a deficit in erythroblast formation. Deletion of Mfrn1 in hepatocytes has no phenotype or biochemical effect under normal conditions. In the presence of increased porphyrin synthesis, however, deletion of Mfrn1 in hepatocytes results in a decreased ability to convert protoporphyrin IX into heme, leading to protoporphyria, cholestasis, and bridging cirrhosis. Our results show that the activity of mitoferrin1 is required to manage an increase in heme synthesis. The data also show that alterations in heme synthesis within hepatocytes can lead to protoporphyria and hepatotoxicity.
Asunto(s)
Anemia/etiología , Proteínas de Transporte de Membrana/deficiencia , Proteínas de Transporte de Membrana/genética , Protoporfiria Eritropoyética/etiología , Anemia/genética , Animales , Secuencia de Bases , Cartilla de ADN/genética , Pérdida del Embrión/genética , Femenino , Marcación de Gen , Hemo/biosíntesis , Hepatocitos/metabolismo , Hierro/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Embarazo , Protoporfiria Eritropoyética/genética , Protoporfirinas/metabolismoRESUMEN
RATIONALE: Netrin-4 regulates vascular development. Identity of netrin-4 endothelial receptor and its subsequent cell functions is controversial. We previously demonstrated that the inhibition of netrin-1 canonical receptors, Unc5B and neogenin, expressed by lymphatic endothelial cells, do not suppress netrin-4-induced cell signaling and functions. Netrin family members were shown to signal through a range of receptors, including integrins (such as α3ß1, α6ß1, and α6ß4) in nonendothelial cells. OBJECTIVE: We tested whether integrins are netrin-4 receptors in the endothelium. METHODS AND RESULTS: The α6ß1 integrin is expressed by endothelial cells, and binds netrin-4 in a dose-dependent manner. Inhibition of α6 or ß1 integrin subunits suppresses netrin-4-induced endothelial cell migration, adhesion, and focal adhesion contact. Netrin-4-stimulated phosphorylation of Src kinase family, effectors of endothelial cell migration, is also abolished by α6 or ß1 inhibition. Finally, netrin-4 and α6ß1 integrin expression colocalize in mouse embryonic, intestine, and tumor vasculature. CONCLUSIONS: The α6ß1 integrin is a netrin-4 receptor in lymphatic endothelium and consequently represents a potential target to inhibit netrin-4-induced metastatic dissemination.
Asunto(s)
Células Endoteliales/metabolismo , Integrina alfa6beta1/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Animales , Vasos Sanguíneos/embriología , Vasos Sanguíneos/metabolismo , Neoplasias de la Mama/irrigación sanguínea , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Femenino , Adhesiones Focales/metabolismo , Humanos , Integrina alfa6beta1/genética , Intestinos/irrigación sanguínea , Vasos Linfáticos/metabolismo , Ratones , Netrinas , Fosforilación , Unión Proteica , Interferencia de ARN , Proteínas Recombinantes/metabolismo , Transfección , Familia-src Quinasas/metabolismoRESUMEN
Netrin-4, a laminin-related secreted protein is an axon guidance cue recently shown essential outside of the nervous system, regulating mammary and lung morphogenesis as well as blood vascular development. Here, we show that Netrin-4, at physiologic doses, induces proliferation, migration, adhesion, tube formation and survival of human lymphatic endothelial cells in vitro comparable to well-characterized lymphangiogenic factors fibroblast growth factor-2 (FGF-2), hepatocyte growth factor (HGF), vascular endothelial growth factor-A (VEGF-A), and vascular endothelial growth factor-C (VEGF-C). Netrin-4 stimulates phosphorylation of intracellular signaling components Akt, Erk and S6, and their specific inhibition antagonizes Netrin-4-induced proliferation. Although Netrin receptors Unc5B and neogenin, are expressed by human lymphatic endothelial cells, suppression of either or both does not suppress Netrin-4-promoted in vitro effects. In vivo, Netrin-4 induces growth of lymphatic and blood vessels in the skin of transgenic mice and in breast tumors. Its overexpression in human and mouse mammary carcinoma cancer cells leads to enhanced metastasis. Finally, Netrin-4 stimulates in vitro and in vivo lymphatic permeability by activating small GTPases and Src family kinases/FAK, and down-regulating tight junction proteins. Together, these data provide evidence that Netrin-4 is a lymphangiogenic factor contributing to tumor dissemination and represents a potential target to inhibit metastasis formation.
Asunto(s)
Células Endoteliales/citología , Linfangiogénesis , Vasos Linfáticos/citología , Factores de Crecimiento Nervioso/metabolismo , Animales , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Células Endoteliales/metabolismo , Femenino , Regulación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Vasos Linfáticos/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Factores de Crecimiento Nervioso/genética , Receptores de Netrina , Netrinas , Receptores de Superficie Celular/metabolismo , Piel/metabolismoRESUMEN
BACKGROUND: ARF nucleotide-binding site opener (ARNO) is a guanine nucleotide-exchange factor for ADP-ribosylation factor proteins. ARF nucleotide-binding site opener also binds MyD88, and small-molecule inhibition of ARNO reduces inflammation in animal models of inflammatory arthritis and acute inflammation. However, whether genetic deletion of Arno in mice reduces pathologic inflammation has not yet been reported. Furthermore, its role in the nasal cavity has yet to be investigated. OBJECTIVE: To generate Arno knockout mice and to determine whether genetic loss of ARNO reduces eosinophilic inflammation in the ovalbumin (OVA) murine model of rhinitis. METHODS: Arno knockout mice were generated and wild type and knockout littermates were subjected to the OVA-induced mouse model of rhinosinutitis. Eosinophilic inflammation was assessed through immunofluorescent quantification of EMBP+ eosinophils in the septal mucosa and cytokine expression was assessed by quantitative polymerase chain reaction. RESULTS: Arno knockout mice are viable and fertile without any noted deficits. Arno wild type and knockout mice subjected to the OVA-induced model of rhinitis demonstrated an average of 314.5 and 153.8 EMBP+ cells per mm2 septal tissue, respectively (P < .05). Goblet cells per mm of basal lamina were assessed via Alcian blue and there was no statistically significant difference between Arno wild type and knockout mice. Ovalbumin-induced expression of interleukin-5 (IL-5) was significantly reduced in Arno knockout mice (P < .05). There was no statistically significant reduction in IL-4, IL-13, or eotaxin-1 expression. CONCLUSIONS: These data demonstrate that deletion of Arno reduces eosinophilic inflammation and IL-5 expression in an OVA-induced model of rhinitis.
Asunto(s)
Interleucina-5 , Rinitis , Animales , Modelos Animales de Enfermedad , Proteínas Activadoras de GTPasa , Inflamación/genética , Interleucina-5/genética , Interleucina-5/metabolismo , Ratones , Ratones Noqueados , Rinitis/genéticaRESUMEN
Breakdown of the blood-central nervous system barrier (BCNSB) is a hallmark of many neuroinflammatory disorders, such as multiple sclerosis (MS). Using a mouse model of MS, experimental autoimmune encephalomyelitis (EAE), we show that endothelial-to-mesenchymal transition (EndoMT) occurs in the CNS before the onset of clinical symptoms and plays a major role in the breakdown of BCNSB function. EndoMT can be induced by an IL-1ß-stimulated signaling pathway in which activation of the small GTPase ADP ribosylation factor 6 (ARF6) leads to crosstalk with the activin receptor-like kinase (ALK)-SMAD1/5 pathway. Inhibiting the activation of ARF6 both prevents and reverses EndoMT, stabilizes BCNSB function, reduces demyelination, and attenuates symptoms even after the establishment of severe EAE, without immunocompromising the host. Pan-inhibition of ALKs also reduces disease severity in the EAE model. Therefore, multiple components of the IL-1ß-ARF6-ALK-SMAD1/5 pathway could be targeted for the treatment of a variety of neuroinflammatory disorders.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Proteínas de Unión al GTP Monoméricas , Esclerosis Múltiple , Receptores de Activinas/metabolismo , Animales , Sistema Nervioso Central/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas de Unión al GTP Monoméricas/metabolismo , Enfermedades Neuroinflamatorias , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de SeñalRESUMEN
Fibroblast growth factors (FGFs) are secreted molecules that activate the RAS/mitogen-activated protein kinase (MAPK) signaling pathway. In zebrafish development, FGF signaling is responsible for establishing dorsal polarity, maintaining the isthmic organizer, and cardiac ventricle formation. Because several ETS factors are known transcriptional mediators of MAPK signaling, we hypothesized that these factors function to mediate FGF signaling processes. In zebrafish, the simultaneous knock-down of three Pea3 ETS proteins, Etv5, Erm, and Pea3, produced phenotypes reminiscent of embryos deficient in FGF signaling. Morphant embryos displayed both cardiac and left/right patterning defects as well as disruption of the isthmic organizer. Furthermore, the expression of FGF target genes was abolished in Pea3 ETS depleted embryos. To understand how FGF signaling and ETS factors control gene expression, transcriptional regulation of dusp6 was studied in mouse and zebrafish. Conserved Pea3 ETS binding sites were identified within the Dusp6 promoter, and reporter assays showed that one of these sites is required for dusp6 induction by FGFs. We further demonstrated the interaction of Pea3 ETS factors with the Dusp6 promoter both in vitro and in vivo. These results revealed the requirement of ETS factors in transducing FGF signals in developmental processes.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Proto-Oncogénicas c-ets/metabolismo , Factores de Transcripción/metabolismo , Pez Cebra/metabolismo , Animales , Sitios de Unión/genética , Diferenciación Celular/genética , Factores de Crecimiento de Fibroblastos/genética , Expresión Génica , Regulación de la Expresión Génica , Ratones , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Oncogenes , Proteínas Proto-Oncogénicas c-ets/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Factores de Transcripción/genética , Pez Cebra/genéticaRESUMEN
Many techniques are currently used in an attempt to regenerate cartilage surfaces in the presence of a chondral or osteochondral defect. Clinical results have been mixed and no single treatment has emerged as being superior. This article reviews the techniques previously and currently being used and evidence to support their use.
Asunto(s)
Cartílago/lesiones , Cartílago/cirugía , Traumatismos de la Rodilla/cirugía , Procedimientos Ortopédicos/métodos , Artroscopía , Condrocitos/trasplante , Humanos , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
AIMS: To investigate factors that contribute to patient decisions regarding attendance for arthroplasty during the COVID-19 pandemic. METHODS: A postal questionnaire was distributed to patients on the waiting list for hip or knee arthroplasty in a single tertiary centre within the UK. Patient factors that may have influenced the decision to attend for arthroplasty, global quality of life (QoL) (EuroQol five-dimension three-level (EQ-5D-3L)), and joint-specific QoL (Oxford Hip or Knee Score) were assessed. Patients were asked at which 'COVID-alert' level they would be willing to attend an NHS and a "COVID-light" hospital for arthroplasty. Independent predictors were assessed using multivariate logistic regression. RESULTS: Of 540 distributed questionnaires, 400 (74.1%; 236 awaiting hip arthroplasty, 164 awaiting knee arthroplasty) complete responses were received and included. Less than half (48.2%) were willing to attend for hip or knee arthroplasty while a UK COVID-19 epidemic was in circulation (COVID-alert levels 3 to 5). Patients with worse joint-specific QoL had a preference to proceed with surgery at COVID-alert levels 3 to 5 compared to levels 1 and 2 (hip arthroplasty odds ratio (OR) 1.54 (95% confidence interval (CI) 1.45 to 1.63); knee arthroplasty OR 1.16 (1.07 to 1.26)). The odds of patients with worse joint-specific QoL being willing to attend for surgery at COVID-alert levels 3 to 5 increased further if surgery in a private, "COVID-light" hospital was available (hip arthroplasty OR 3.50 (95% CI 3.26 to 3.71); knee arthroplasty OR 1.41 (95% CI 1.29 to 1.53). CONCLUSION: Patient decisions surrounding elective surgery have been influenced by the global COVID-19 pandemic, highlighting the importance of patient involvement in ensuring optimized provision of elective surgery during these challenging times. Cite this article: Bone Jt Open 2021;2(4):261-270.
RESUMEN
The establishment of anteroposterior (AP) polarity in the early mouse epiblast is crucial for the initiation of gastrulation and the subsequent formation of the embryonic (head to tail) axis. The localization of anterior and posterior determining genes to the appropriate region of the embryo is a dynamic process that underlies this early polarity. Several studies indicate that morphological and molecular markers which define the early AP axis are first aligned along the short axis of the elliptical egg cylinder. Subsequently, just prior to the time of primitive streak formation, a conformational change in the embryo realigns these markers with the long axis. We demonstrate that embryos lacking the signaling factor Wnt3 exhibit defects in this axial realignment. In addition, chimeric analyses and conditional removal of Wnt3 activity reveal that Wnt3 expression in the epiblast is required for induction of the primitive streak and mesoderm whereas activity in the posterior visceral endoderm is dispensable.
Asunto(s)
Tipificación del Cuerpo , Estratos Germinativos/embriología , Estratos Germinativos/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , Alelos , Animales , Quimera , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario , Gástrula/metabolismo , Integrasas/metabolismo , Ratones , Modelos Biológicos , Mutación/genética , Proteína Wnt3RESUMEN
Stimulation of white adipose tissue (WAT) browning is considered as a potential approach to treat obesity and metabolic diseases. Our previous studies have shown that phytochemical butein can stimulate WAT browning through induction of Prdm4 in adipocytes. Here, we investigated the effects of butein on diet-induced obesity and its underlying molecular mechanism. Treatment with butein prevented weight gains and improved metabolic profiles in diet-induced obese mice. Butein treatment groups also displayed higher body temperature, increased energy expenditure, and enhanced expression of thermogenic genes in adipose tissue. Butein also suppressed body weight gains and improved glucose and insulin tolerance in mice housed at thermoneutrality (30 °C). These effects were associated with adipose-selective induction of Prdm4, suggesting the role of Prdm4 in butein-mediated anti-obese effects. To directly assess the in vivo role of Prdm4, we generated aP2-Prdm4 transgenic mouse lines overexpressing Prdm4 in adipose tissues. Adipose-specific transgenic expression of Prdm4 recapitulated the butein's actions in stimulating energy expenditure, cold tolerance, and thermogenic gene expression, resulting in prevention of obesity and improvement of metabolism. Mechanistically, direct inhibition of PI3Kα activity followed by selective suppression of its downstream Akt1 mirrored butein's effect on Ucp1 expression and oxygen consumption. In addition, effects of butein were completely abolished in Akt1 KO mouse embryonic fibroblasts. Together, these studies demonstrate the role of butein in obesity and metabolic diseases, further highlighting that adipose PI3Kα-Akt1-Prdm4 axis is a regulator of energy expenditure.
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Tejido Adiposo/metabolismo , Proteínas de Unión al ADN/metabolismo , Metabolismo Energético/fisiología , Resistencia a la Insulina/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción/metabolismo , Aumento de Peso/fisiología , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Adipocitos/fisiología , Tejido Adiposo/efectos de los fármacos , Animales , Línea Celular , Chalconas/farmacología , Dieta Alta en Grasa/efectos adversos , Metabolismo Energético/efectos de los fármacos , Ratones , Ratones Noqueados , Ratones Obesos , Obesidad/metabolismo , Termogénesis/efectos de los fármacos , Termogénesis/fisiología , Proteína Desacopladora 1/metabolismo , Aumento de Peso/efectos de los fármacosRESUMEN
The devastating sequelae of diabetes mellitus include microvascular permeability, which results in retinopathy. Despite clinical and scientific advances, there remains a need for new approaches to treat retinopathy. Here, we have presented a possible treatment strategy, whereby targeting the small GTPase ARF6 alters VEGFR2 trafficking and reverses signs of pathology in 4 animal models that represent features of diabetic retinopathy and in a fifth model of ocular pathological angiogenesis. Specifically, we determined that the same signaling pathway utilizes distinct GEFs to sequentially activate ARF6, and these GEFs exert distinct but complementary effects on VEGFR2 trafficking and signal transduction. ARF6 activation was independently regulated by 2 different ARF GEFs - ARNO and GEP100. Interaction between VEGFR2 and ARNO activated ARF6 and stimulated VEGFR2 internalization, whereas a VEGFR2 interaction with GEP100 activated ARF6 to promote VEGFR2 recycling via coreceptor binding. Intervening in either pathway inhibited VEGFR2 signal output. Finally, using a combination of in vitro, cellular, genetic, and pharmacologic techniques, we demonstrated that ARF6 is pivotal in VEGFR2 trafficking and that targeting ARF6-mediated VEGFR2 trafficking has potential as a therapeutic approach for retinal vascular diseases such as diabetic retinopathy.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Retinopatía Diabética/metabolismo , Transducción de Señal , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Línea Celular , Retinopatía Diabética/genética , Retinopatía Diabética/patología , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Humanos , Transporte de Proteínas , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
BACKGROUND: An investigation of matrix metalloproteinase-9 (MMP-9) and its influence on vascular invasion in the secondary ossification center at the chondroepiphysis of developing long bones was undertaken. The effect of MMP-9 was compared with that of basic fibroblast growth factor (b-FGF), a potent angiogenic factor, and we assessed the chorioallantoic membrane (CAM) culture as a model for angiogenesis in osteochondral tissue. METHODS: Seventy-two femoral and seventy-two humeral heads of thirty-six four-day postnatal rabbits were dissected immediately after each animal was killed. Solutions of MMP-9, b-FGF, and phosphate-buffered saline solution were applied, and the femoral and humeral chondroepiphyseal explants were incubated for ten days in CAM culture. This was used as an in vivo model to investigate the growth of blood vessels into the femoral and humeral heads of the neonatal rabbit. The explants were harvested from the CAM culture and analyzed histologically. A three-day incubation was also performed to look for early signs of vascular ingrowth into the cartilage matrix. RESULTS: One hundred and twenty epiphyses from thirty rabbits were placed onto CAM culture successfully; of these, two were harvested at three days to assess early changes and 118 were harvested at ten days. Forty of the 118 cultures were still viable when harvested after ten days, giving a 33% yield. Both MMP-9 and b-FGF caused an increased vascular invasion into the chondroepiphysis. New blood vessels derived from the chorioallantoic membrane within cartilage canals were more numerous in MMP-9 treated epiphyses, and larger canals were more commonly seen when compared with a control group. CONCLUSIONS: These findings confirmed that b-FGF is angiogenic at the chondroepiphysis. Matrix metalloproteinase-9 appears to be implicated in vascular invasion and induces the formation of new cartilage canals at the chondroepiphysis. The CAM culture model was a useful model for investigating angiogenesis in osteochondral tissue. CLINICAL RELEVANCE: This study adds to the understanding of the complex biochemical interaction that occurs in cartilage when the advancing vasculature begins growing into the chondroepiphysis. A better knowledge of this angiogenic process will enable a better understanding of the pathological failure or disturbance of vasculogenesis, which results in dysplastic growth disorders and osteonecrosis.
Asunto(s)
Condrogénesis/efectos de los fármacos , Fémur/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Húmero/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/farmacología , Osteogénesis/efectos de los fármacos , Animales , Animales Recién Nacidos , Epífisis/efectos de los fármacos , Epífisis/crecimiento & desarrollo , Fémur/crecimiento & desarrollo , Húmero/crecimiento & desarrollo , Conejos , Técnicas de Cultivo de TejidosRESUMEN
Activating mutations in Gαq proteins, which form the α subunit of certain heterotrimeric G proteins, drive uveal melanoma oncogenesis by triggering multiple downstream signaling pathways, including PLC/PKC, Rho/Rac, and YAP. Here we show that the small GTPase ARF6 acts as a proximal node of oncogenic Gαq signaling to induce all of these downstream pathways as well as ß-catenin signaling. ARF6 activates these diverse pathways through a common mechanism: the trafficking of GNAQ and ß-catenin from the plasma membrane to cytoplasmic vesicles and the nucleus, respectively. Blocking ARF6 with a small-molecule inhibitor reduces uveal melanoma cell proliferation and tumorigenesis in a mouse model, confirming the functional relevance of this pathway and suggesting a therapeutic strategy for Gα-mediated diseases.
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Factores de Ribosilacion-ADP/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Melanoma/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Neoplasias de la Úvea/tratamiento farmacológico , beta Catenina/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/antagonistas & inhibidores , Factores de Ribosilacion-ADP/genética , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Citoplasma/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Humanos , Melanoma/genética , Melanoma/metabolismo , Ratones , Trasplante de Neoplasias , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/metabolismoRESUMEN
The lack of readily available, patient-derived materials for molecular genetic testing of many heterozygous or rare disorders creates a major impediment for laboratory proficiency and quality control procedures. The paucity of clinically derived mutation-positive samples could be surmounted if it were possible to construct artificial samples containing mutations of interest that would sufficiently resemble natural human samples. Such samples could then function as acceptable and realistic performance evaluation challenges and quality control reagents for recipient laboratories. Using the cystic fibrosis gene (CFTR) as a prototype, we have devised and executed experiments designed to generate unique DNA samples that could be used for these purposes. We used site-directed mutagenesis to generate mutations of interest in plasmid DNA derived from common bacterial artificial chromosome sources containing the cystic fibrosis transmembrane conductance receptor gene. CFTR mutations G85E and 1078delT were chosen to represent mutations in the original American College of Medical Genetics-recommended population-screening panel of 25 mutations. DNA samples containing predetermined concentrations and ratios of wild-type and mutated plasmids, bacterial artificial chromosomes of interest, and nonhuman genomic carrier DNA were characterized and tested in-house and in a group of nine pilot testing laboratories using a variety of technical platforms. The results indicate that these constructs, containing CFTR mutations in heterozygous and homozygous states, can serve as valid and accessible materials for quality assurance, including performance evaluation, proficiency testing, and assay quality control.
Asunto(s)
Análisis Mutacional de ADN/métodos , Análisis Mutacional de ADN/normas , Pruebas Genéticas/normas , Mutagénesis Sitio-Dirigida , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Mutación , Plásmidos/genética , Control de CalidadRESUMEN
BACKGROUND AND PURPOSE: Ablation by cold (cryoablation) or radiofrequency energy (RFA), has been popularized for the treatment of small renal tumors. Regrettably, there currently is no reliable method of radiologically monitoring the propagation of RF lesions in real time. Ultrasonography enhanced by gas-filled microbubble contrast agents allows depiction of regions of tissue perfusion and has been described as a useful adjunct in diagnosing renal pseudotumors, improving prostate biopsy results, and confirming successful ablation of liver tumors. We hypothesized that contrast-enhanced ultrasonography (CEUS) would allow us to define, in real time, areas of cell death secondary to RFA and thus determine successful treatment. MATERIALS AND METHODS: Five female swine underwent initial laparoscopic exploration and creation of ipsilateral upper- and lower-pole renal RFA lesions. Lesion size was measured with standard gray-scale, Doppler, and microbubble CEUS. After 2 weeks, an identical procedure was performed on the contralateral kidney, including repeat sonographic measurements on the first kidney. All swine were then immediately sacrificed, and both kidneys (20 lesions) were harvested for pathologic analysis (hematoxylin-eosin and nicotinamide adenine dinucleotide stains). Radiographic lesion size was then compared with the gross and microscopic findings. RESULTS: The RFA lesions could not be imaged accurately in real time with standard gray-scale or Doppler sonography. However, microbubble CEUS was able to monitor parenchymal blood flow and, thus, the lesions (no blood flow) in real time. Hypoechoic lesions (no bubble enhancement) imaged during contrast sonography corresponded with regions of cell death as demonstrated on pathologic analysis. CONCLUSIONS: Microbubble CEUS is can monitor RFA lesions in real time. This novel imaging modality should allow more effective renal tumor ablation.
Asunto(s)
Ablación por Catéter , Riñón/cirugía , Monitoreo Intraoperatorio/instrumentación , Monitoreo Intraoperatorio/métodos , Ultrasonografía , Animales , Sistemas de Computación , Femenino , Riñón/diagnóstico por imagen , Neoplasias Renales/diagnóstico por imagen , Neoplasias Renales/cirugía , Modelos Animales , Porcinos , Procedimientos Quirúrgicos Urológicos/instrumentación , Procedimientos Quirúrgicos Urológicos/métodosRESUMEN
Vitamin D is a known modulator of inflammation. Native dietary vitamin D3 is thought to be bio-inactive, and beneficial vitamin D3 effects are thought to be largely mediated by the metabolite 1,25(OH)2D3. Reduced serum levels of the most commonly measured precursor metabolite, 25(OH)D3, is linked to an increased risk of multiple inflammatory diseases, including: cardiovascular disease, arthritis, multiple sclerosis, and sepsis. Common to all of these diseases is the disruption of endothelial stability and an enhancement of vascular leak. We previously performed an unbiased chemical suppressor screen on a genetic model of vascular instability, and identified cholecalciferol (D3, dietary Vitamin D3) as a factor that had profound and immediate stabilizing and therapeutic effects in that model. In this manuscript we show that the presumed inactive sterol, D3, is actually a potent and general mediator of endothelial stability at physiologically relevant concentrations. We further demonstrate that this phenomenon is apparent in vitamin D3 metabolites 25(OH)D3 and 1,25(OH)2D3, and that the effects are independent of the canonical transcription-mediated vitamin D pathway. Our data suggests the presence of an alternative signaling modality by which D3 acts directly on endothelial cells to prevent vascular leak. The finding that D3 and its metabolites modulate endothelial stability may help explain the clinical correlations between low serum vitamin D levels and the many human diseases with well-described vascular dysfunction phenotypes.