RESUMEN
Connexin37-mediated regulation of cell cycle modulators and, consequently, growth arrest lack mechanistic understanding. We previously showed that arterial shear stress up-regulates Cx37 in endothelial cells and activates a Notch/Cx37/p27 signaling axis to promote G1 cell cycle arrest, and this is required to enable arterial gene expression. However, how induced expression of a gap junction protein, Cx37, up-regulates cyclin-dependent kinase inhibitor p27 to enable endothelial growth suppression and arterial specification is unclear. Herein, we fill this knowledge gap by expressing wild-type and regulatory domain mutants of Cx37 in cultured endothelial cells expressing the Fucci cell cycle reporter. We determined that both the channel-forming and cytoplasmic tail domains of Cx37 are required for p27 up-regulation and late G1 arrest. Mechanistically, the cytoplasmic tail domain of Cx37 interacts with, and sequesters, activated ERK in the cytoplasm. This then stabilizes pERK nuclear target Foxo3a, which up-regulates p27 transcription. Consistent with previous studies, we found this Cx37/pERK/Foxo3a/p27 signaling axis functions downstream of arterial shear stress to promote endothelial late G1 state and enable up-regulation of arterial genes.
Asunto(s)
Conexinas , Células Endoteliales , Células Endoteliales/metabolismo , Puntos de Control del Ciclo Celular/genética , Conexinas/genética , Conexinas/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular , Núcleo Celular/metabolismo , Proteína alfa-4 de Unión ComunicanteRESUMEN
The subventricular zone (SVZ) is the largest neural stem cell (NSC) niche in the adult brain; herein, the blood-brain barrier is leaky, allowing direct interactions between NSCs and endothelial cells (ECs). Mechanisms by which direct NSC-EC interactions in the adult SVZ control NSC behavior are unclear. We found that Cx43 is highly expressed by SVZ NSCs and ECs, and its deletion in either leads to increased NSC proliferation and neuroblast generation, suggesting that Cx43-mediated NSC-EC interactions maintain NSC quiescence. This is further supported by single-cell RNA sequencing and in vitro studies showing that ECs control NSC proliferation by regulating expression of genes associated with NSC quiescence and/or activation in a Cx43-dependent manner. Cx43 mediates these effects in a channel-independent manner involving its cytoplasmic tail and ERK activation. Such insights inform adult NSC regulation and maintenance aimed at stem cell therapies for neurodegenerative disorders.
Asunto(s)
Conexina 43 , Ventrículos Laterales , Células Endoteliales/metabolismo , Encéfalo/metabolismo , Neurogénesis/fisiologíaRESUMEN
During blood vessel development, endothelial cells become specified toward arterial or venous fates to generate a circulatory network that provides nutrients and oxygen to, and removes metabolic waste from, all tissues. Arterial-venous specification occurs in conjunction with suppression of endothelial cell cycle progression; however, the mechanistic role of cell cycle state is unknown. Herein, using Cdh5-CreERT2;R26FUCCI2aR reporter mice, we find that venous endothelial cells are enriched for the FUCCI-Negative state (early G1) and BMP signaling, while arterial endothelial cells are enriched for the FUCCI-Red state (late G1) and TGF-ß signaling. Furthermore, early G1 state is essential for BMP4-induced venous gene expression, whereas late G1 state is essential for TGF-ß1-induced arterial gene expression. Pharmacologically induced cell cycle arrest prevents arterial-venous specification defects in mice with endothelial hyperproliferation. Collectively, our results show that distinct endothelial cell cycle states provide distinct windows of opportunity for the molecular induction of arterial vs. venous fate.
Asunto(s)
Células Endoteliales , Factor de Crecimiento Transformador beta1 , Animales , Arterias/metabolismo , Ciclo Celular , Células Endoteliales/metabolismo , Ratones , Oxígeno/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , VenasRESUMEN
Development of blood-forming (hemogenic) endothelial cells that give rise to hematopoietic stem and progenitor cells (HSPCs) is critical during embryogenesis to generate the embryonic and postnatal hematopoietic system. We previously demonstrated that the specification of murine hemogenic endothelial cells is promoted by retinoic acid (RA) signaling and requires downstream endothelial cell cycle control. Whether this mechanism is conserved in human hemogenic endothelial cell specification is unknown. Here, we present a protocol to derive primordial endothelial cells from human embryonic stem cells and promote their specification toward hemogenic endothelial cells. Furthermore, we demonstrate that RA treatment significantly increases human hemogenic endothelial cell specification. That is, RA promotes endothelial cell cycle arrest to enable RA-induced instructive signals to upregulate the genes needed for hematopoietic transition. These insights provide guidance for the ex vivo generation of autologous human hemogenic endothelial cells that are needed to produce human HSPCs for regenerative medicine applications.
Asunto(s)
Ciclo Celular/genética , Células Endoteliales/metabolismo , Tretinoina/metabolismo , Animales , Diferenciación Celular , Humanos , RatonesRESUMEN
The vascular barrier that separates blood from tissues is actively regulated by the endothelium and is essential for transport, inflammation, and haemostasis. Haemodynamic shear stress plays a critical role in maintaining endothelial barrier function, but how this occurs remains unknown. Here we use an engineered organotypic model of perfused microvessels to show that activation of the transmembrane receptor NOTCH1 directly regulates vascular barrier function through a non-canonical, transcription-independent signalling mechanism that drives assembly of adherens junctions, and confirm these findings in mouse models. Shear stress triggers DLL4-dependent proteolytic activation of NOTCH1 to expose the transmembrane domain of NOTCH1. This domain mediates establishment of the endothelial barrier; expression of the transmembrane domain of NOTCH1 is sufficient to rescue defects in barrier function induced by knockout of NOTCH1. The transmembrane domain restores barrier function by catalysing the formation of a receptor complex in the plasma membrane consisting of vascular endothelial cadherin, the transmembrane protein tyrosine phosphatase LAR, and the RAC1 guanidine-exchange factor TRIO. This complex activates RAC1 to drive assembly of adherens junctions and establish barrier function. Canonical transcriptional signalling via Notch is highly conserved in metazoans and is required for many processes in vascular development, including arterial-venous differentiation, angiogenesis and remodelling. We establish the existence of a non-canonical cortical NOTCH1 signalling pathway that regulates vascular barrier function, and thus provide a mechanism by which a single receptor might link transcriptional programs with adhesive and cytoskeletal remodelling.
Asunto(s)
Uniones Adherentes/metabolismo , Endotelio Vascular/metabolismo , Complejos Multiproteicos/metabolismo , Receptor Notch1/metabolismo , Uniones Adherentes/enzimología , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Línea Celular , Endotelio Vascular/enzimología , Femenino , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Ratones , Complejos Multiproteicos/química , Fosfoproteínas/metabolismo , Dominios Proteicos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Receptor Notch1/química , Transducción de Señal , Proteínas de Unión al GTP rac/metabolismoRESUMEN
OBJECTIVE: The calcium composition of atherosclerotic plaque is thought to be associated with increased risk for cardiovascular events, but whether plaque calcium itself is predictive of worsening clinical outcomes remains highly controversial. Inflammation is likely a key mediator of vascular calcification, but immune signaling mechanisms that promote this process are minimally understood. APPROACH AND RESULTS: Here, we identify Rac2 as a major inflammatory regulator of signaling that directs plaque osteogenesis. In experimental atherogenesis, Rac2 prevented progressive calcification through its suppression of Rac1-dependent macrophage interleukin-1ß (IL-1ß) expression, which in turn is a key driver of vascular smooth muscle cell calcium deposition by its ability to promote osteogenic transcriptional programs. Calcified coronary arteries from patients revealed decreased Rac2 expression but increased IL-1ß expression, and high coronary calcium burden in patients with coronary artery disease was associated with significantly increased serum IL-1ß levels. Moreover, we found that elevated IL-1ß was an independent predictor of cardiovascular death in those subjects with high coronary calcium burden. CONCLUSIONS: Overall, these studies identify a novel Rac2-mediated regulation of macrophage IL-1ß expression, which has the potential to serve as a powerful biomarker and therapeutic target for atherosclerosis.
Asunto(s)
Enfermedades de la Aorta/enzimología , Aterosclerosis/enzimología , Enfermedad de la Arteria Coronaria/enzimología , Mediadores de Inflamación/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/enzimología , Placa Aterosclerótica , Calcificación Vascular/enzimología , Proteínas de Unión al GTP rac/metabolismo , Animales , Aorta/enzimología , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/prevención & control , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/patología , Aterosclerosis/prevención & control , Células Cultivadas , Enfermedad de la Arteria Coronaria/mortalidad , Enfermedad de la Arteria Coronaria/patología , Vasos Coronarios/enzimología , Vasos Coronarios/patología , Femenino , Predisposición Genética a la Enfermedad , Humanos , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Macrófagos/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/enzimología , Miocitos del Músculo Liso/patología , Neuropéptidos/metabolismo , Fenotipo , Pronóstico , Transducción de Señal , Transfección , Regulación hacia Arriba , Calcificación Vascular/mortalidad , Calcificación Vascular/patología , Proteínas de Unión al GTP rac/deficiencia , Proteínas de Unión al GTP rac/genética , Proteína de Unión al GTP rac1/metabolismo , Proteína RCA2 de Unión a GTPRESUMEN
Hematopoietic stem cells (HSCs) reside within a specialized niche where interactions with vasculature, osteoblasts, and stromal components regulate their self-renewal and differentiation. Little is known about bone marrow niche formation or the role of its cellular components in HSC development; therefore, we established the timing of murine fetal long bone vascularization and ossification relative to the onset of HSC activity. Adult-repopulating HSCs emerged at embryonic day 16.5 (E16.5), coincident with marrow vascularization, and were contained within the c-Kit(+)Sca-1(+)Lin(-) (KSL) population. We used Osterix-null (Osx(-/-)) mice that form vascularized marrow but lack osteolineage cells to dissect the role(s) of these cellular components in HSC development. Osx(-/-) fetal bone marrow cells formed multilineage colonies in vitro but were hyperproliferative and failed to home to and/or engraft transplant recipients. Thus, in developing bone marrow, the vasculature can sustain multilineage progenitors, but interactions with osteolineage cells are needed to regulate long-term HSC proliferation and potential.
Asunto(s)
Médula Ósea/embriología , Células Madre Embrionarias/citología , Células Madre Hematopoyéticas/citología , Osteogénesis , Nicho de Células Madre , Animales , Médula Ósea/irrigación sanguínea , Linaje de la Célula , Proliferación Celular , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/fisiología , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/fisiología , Ratones , Ratones Endogámicos C57BL , Neovascularización Fisiológica , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Factor de Transcripción Sp7 , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Delineating the mechanism or mechanisms that regulate the specification of hemogenic endothelial cells from primordial endothelium is critical for optimizing their derivation from human stem cells for clinical therapies. We previously determined that retinoic acid (RA) is required for hemogenic specification, as well as cell-cycle control, of endothelium during embryogenesis. Herein, we define the molecular signals downstream of RA that regulate hemogenic endothelial cell development and demonstrate that cell-cycle control is required for this process. We found that re-expression of c-Kit in RA-deficient (Raldh2(-/-)) primordial endothelium induced Notch signaling and p27 expression, which restored cell-cycle control and rescued hemogenic endothelial cell specification and function. Re-expression of p27 in RA-deficient and Notch-inactivated primordial endothelial cells was sufficient to correct their defects in cell-cycle regulation and hemogenic endothelial cell development. Thus, RA regulation of hemogenic endothelial cell specification requires c-Kit, notch signaling, and p27-mediated cell-cycle control.
Asunto(s)
Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Células Endoteliales/metabolismo , Células Madre Hematopoyéticas/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptor Notch1/metabolismo , Transducción de Señal/fisiología , Aldehído Oxidorreductasas/genética , Animales , Antineoplásicos/farmacología , Puntos de Control del Ciclo Celular/fisiología , Diferenciación Celular/fisiología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Técnicas de Cultivo de Embriones , Células Endoteliales/citología , Femenino , Células Madre Hematopoyéticas/citología , Operón Lac , Lentivirus/genética , Masculino , Ratones , Ratones Noqueados , Embarazo , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-myb/genética , Transducción de Señal/efectos de los fármacos , Tretinoina/farmacologíaRESUMEN
Asthma, the prototypic Th2-mediated inflammatory disorder of the lung, is an emergent disease worldwide. Vascular endothelial growth factor (VEGF) is a critical regulator of pulmonary Th2 inflammation, but the underlying mechanism and the roles of microRNAs (miRNAs) in this process have not been defined. Here we show that lung-specific overexpression of VEGF decreases miR-1 expression in the lung, most prominently in the endothelium, and a similar down-regulation occurs in lung endothelium in Th2 inflammation models. Intranasal delivery of miR-1 inhibited inflammatory responses to ovalbumin, house dust mite, and IL-13 overexpression. Blocking VEGF inhibited Th2-mediated lung inflammation, and this was restored by antagonizing miR-1. Using mRNA arrays, Argonaute pull-down assays, luciferase expression assays, and mutational analysis, we identified Mpl as a direct target of miR-1 and showed that VEGF controls the expression of endothelial Mpl during Th2 inflammation via the regulation of miR-1. In vivo knockdown of Mpl inhibited Th2 inflammation and indirectly inhibited the expression of P-selectin in lung endothelium. These experiments define a novel VEGF-miR-1-Mpl-P-selectin effector pathway in lung Th2 inflammation and herald the utility of miR-1 and Mpl as potential therapeutic targets for asthma.
Asunto(s)
MicroARNs/genética , Selectina-P/genética , Neumonía/genética , Neumonía/inmunología , Receptores de Trombopoyetina/genética , Células Th2/inmunología , Células Th2/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Regiones no Traducidas 3' , Animales , Endotelio/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Pulmón/inmunología , Pulmón/metabolismo , Ratones , Selectina-P/metabolismo , Interferencia de ARN , Receptores de Trombopoyetina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Like humans with Parkinson's disease (PD), the ak mouse lacks the majority of the substantia nigra pars compacta (SNc) and experiences striatal denervation. The purpose of this study was to test whether motor abnormalities in the ak mouse progress over time, and whether motor function could be associated with temporal alterations in the striatal transcriptome. Ak and wt mice (28 to 180 days old) were tested using paradigms sensitive to nigrostriatal dysfunction. Results were analyzed using a linear mixed model. Ak mice significantly underperformed wt controls in rotarod, balance beam, string test, pole test and cotton shred tests at all ages examined. Motor performance in ak mice remained constant over the first 6 months of life, with the exception of the cotton shred test, in which ak mice exhibited marginal decline in performance. Dorsal striatal semi-quantitative RT-PCR for 19 dopaminergic, cholinergic, glutaminergic and catabolic genes was performed in 1- and 6-month-old groups of ak and wt mice. Preproenkephalin levels in ak mice were elevated in both age groups. Drd1, 3 and 4 levels declined over time, in contrast to increasing Drd2 expression. Additional findings included decreased Chrnalpha6 expression and elevated VGluT1 expression at both time points in ak mice and elevated AchE expression in young ak mice only. Results confirm that motor ability does not decline significantly for the first 6 months of life in ak mice. Their striatal gene expression patterns are consistent with dopaminergic denervation, and change over time, despite relatively unaltered motor performance.