RESUMO
Angiogenesis, the fundamental process by which new blood vessels form from existing ones, depends on precise spatial and temporal gene expression within specific compartments of the endothelium. However, the molecular links between proangiogenic signals and downstream gene expression remain unclear. During sprouting angiogenesis, the specification of endothelial cells into the tip cells that lead new blood vessel sprouts is coordinated by vascular endothelial growth factor A (VEGFA) and Delta-like ligand 4 (Dll4)/Notch signaling and requires high levels of Notch ligand DLL4. Here, we identify MEF2 transcription factors as crucial regulators of sprouting angiogenesis directly downstream from VEGFA. Through the characterization of a Dll4 enhancer directing expression to endothelial cells at the angiogenic front, we found that MEF2 factors directly transcriptionally activate the expression of Dll4 and many other key genes up-regulated during sprouting angiogenesis in both physiological and tumor vascularization. Unlike ETS-mediated regulation, MEF2-binding motifs are not ubiquitous to all endothelial gene enhancers and promoters but are instead overrepresented around genes associated with sprouting angiogenesis. MEF2 target gene activation is directly linked to VEGFA-induced release of repressive histone deacetylases and concurrent recruitment of the histone acetyltransferase EP300 to MEF2 target gene regulatory elements, thus establishing MEF2 factors as the transcriptional effectors of VEGFA signaling during angiogenesis.
Assuntos
Células Endoteliais/citologia , Células Endoteliais/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição MEF2/metabolismo , Neovascularização Fisiológica/genética , Animais , Células Cultivadas , Embrião não Mamífero , Células Endoteliais/enzimologia , Elementos Facilitadores Genéticos/genética , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fatores de Transcrição MEF2/química , Fatores de Transcrição MEF2/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Neovascularização Patológica/genética , Domínios e Motivos de Interação entre Proteínas , Retina/embriologia , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Peixe-ZebraRESUMO
OBJECTIVE: Atherosclerosis predominantly forms in regions of oscillatory shear stress while regions of laminar shear stress are protected. This protection is partly through the endothelium in laminar flow regions expressing an anti-inflammatory and antithrombotic gene expression program. Several molecular pathways transmitting these distinct flow patterns to the endothelium have been defined. Our objective is to define the role of the MEF2 (myocyte enhancer factor 2) family of transcription factors in promoting an atheroprotective endothelium. Approach and Results: Here, we show through endothelial-specific deletion of the 3 MEF2 factors in the endothelium, Mef2a, -c, and -d, that MEF2 is a critical regulator of vascular homeostasis. MEF2 deficiency results in systemic inflammation, hemorrhage, thrombocytopenia, leukocytosis, and rapid lethality. Transcriptome analysis reveals that MEF2 is required for normal regulation of 3 pathways implicated in determining the flow responsiveness of the endothelium. Specifically, MEF2 is required for expression of Klf2 and Klf4, 2 partially redundant factors essential for promoting an anti-inflammatory and antithrombotic endothelium. This critical requirement results in phenotypic similarities between endothelial-specific deletions of Mef2a/c/d and Klf2/4. In addition, MEF2 regulates the expression of Notch family genes, Notch1, Dll1, and Jag1, which also promote an atheroprotective endothelium. In contrast to these atheroprotective pathways, MEF2 deficiency upregulates an atherosclerosis promoting pathway through increasing the amount of TAZ (transcriptional coactivator with PDZ-binding motif). CONCLUSIONS: Our results implicate MEF2 as a critical upstream regulator of several transcription factors responsible for gene expression programs that affect development of atherosclerosis and promote an anti-inflammatory and antithrombotic endothelium. Graphic Abstract: A graphic abstract is available for this article.
Assuntos
Aterosclerose/metabolismo , Endotélio Vascular/metabolismo , Fatores de Transcrição MEF2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Aterosclerose/genética , Aterosclerose/patologia , Endotélio Vascular/patologia , Feminino , Regulação da Expressão Gênica , Homeostase , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição MEF2/deficiência , Fatores de Transcrição MEF2/genética , Masculino , Camundongos , Camundongos Knockout , Receptores Notch/genética , Transdução de Sinais , Transativadores/metabolismoRESUMO
The roles of cellular orientation during trabecular and ventricular wall morphogenesis are unknown, and so are the underlying mechanisms that regulate cellular orientation. Myocardial-specific Numb and Numblike double-knockout (MDKO) hearts display a variety of defects, including in cellular orientation, patterns of mitotic spindle orientation, trabeculation, and ventricular compaction. Furthermore, Numb- and Numblike-null cardiomyocytes exhibit cellular behaviors distinct from those of control cells during trabecular morphogenesis based on single-cell lineage tracing. We investigated how Numb regulates cellular orientation and behaviors and determined that N-cadherin levels and membrane localization are reduced in MDKO hearts. To determine how Numb regulates N-cadherin membrane localization, we generated an mCherry:Numb knockin line and found that Numb localized to diverse endocytic organelles but mainly to the recycling endosome. Consistent with this localization, cardiomyocytes in MDKO did not display defects in N-cadherin internalization but rather in postendocytic recycling to the plasma membrane. Furthermore, N-cadherin overexpression via a mosaic model partially rescued the defects in cellular orientation and trabeculation of MDKO hearts. Our study unravels a phenomenon that cardiomyocytes display spatiotemporal cellular orientation during ventricular wall morphogenesis, and its disruption leads to abnormal trabecular and ventricular wall morphogenesis. Furthermore, we established a mechanism by which Numb modulates cellular orientation and consequently trabecular and ventricular wall morphogenesis by regulating N-cadherin recycling to the plasma membrane.
Assuntos
Caderinas/metabolismo , Ventrículos do Coração/embriologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Organogênese , Animais , Caderinas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Miócitos Cardíacos/citologia , Proteínas do Tecido Nervoso/genéticaRESUMO
PURPOSE: Understanding potential bias due to rarity of the outcome is important when monitoring newly approved drugs and drugs with low availability to the general public. Although there is an increasing use of online surveys to investigate health outcomes, the limits of inference due to drug availability have not been studied. The goal of this study was to quantify the relationship between dispensing of prescription drugs and estimates of use in an online general population survey. METHODS: An online repeated, cross-sectional survey from 2018 to 2020 was used to estimate the number of adults in the United States who used prescription drugs in the general population and compared to estimated number of prescriptions dispensed over an equivalent time period. Joinpoint regression was used to quantify thresholds. A sample of respondents was retested to estimate reliability statistics. RESULTS: A model with a single threshold was the best fit, with the estimated threshold of 565 000 (95% CI: 9500-11 600 000) prescriptions dispensed per year. Above the threshold, there was a significant association between dispensing and estimates (p < 0.001); below the threshold, the relationship was not significant (p = 0.912). Above the threshold, responses were more reliable than random chance, and reliability steadily increased with increased dispensing. CONCLUSIONS: These results suggest the threshold demarcates two distinct pharmacoepidemiological paradigms when investigating drug use in general population surveys. Dispensing can be used as a guide to determine the epidemiological paradigm that is best suited.
Assuntos
Prescrições de Medicamentos , Medicamentos sob Prescrição , Adulto , Estudos Transversais , Humanos , Farmacoepidemiologia , Reprodutibilidade dos Testes , Estados Unidos/epidemiologiaRESUMO
Calcineurin is required for B cell receptor (BCR)-induced proliferation of mature B cells. Paradoxically, loss of NFAT transcription factors, themselves calcineurin targets, induces hyperactivity, which suggests that calcineurin targets other than NFAT are required for BCR-induced proliferation. Here we demonstrate a function for the calcineurin-regulated transcription factor Mef2c in B cells. BCR-induced calcium mobilization was intact after Mef2c deletion, but loss of Mef2c caused defects in B cell proliferation and survival after BCR stimulation in vitro and lower T cell-dependent antibody responses and germinal center formation in vivo. Mef2c activity was specific to BCR stimulation, as Toll-like receptor and CD40 signaling induced normal responses in Mef2c-deficient B cells. Mef2c-dependent targets included the genes encoding cyclin D2 and the prosurvival factor Bcl-x(L). Our results emphasize an unrecognized but critical function for Mef2c in BCR signaling.
Assuntos
Linfócitos B/fisiologia , Calcineurina/metabolismo , Sobrevivência Celular/fisiologia , Fatores de Regulação Miogênica/fisiologia , Receptores de Antígenos de Linfócitos B/fisiologia , Animais , Ciclo Celular/fisiologia , Fatores de Transcrição MEF2 , Camundongos , Fatores de Transcrição/fisiologiaRESUMO
We present a twistorlike formula for the complete tree-level S matrix of six-dimensional (6D) (2,0) supergravity coupled to 21 Abelian tensor multiplets. This is the low-energy effective theory that corresponds to type IIB superstring theory compactified on a K3 surface. The formula is expressed as an integral over the moduli space of certain rational maps of the punctured Riemann sphere. By studying soft limits of the formula, we are able to explore the local moduli space of this theory, {[SO(5,21)]/[SO(5)×SO(21)]}. Finally, by dimensional reduction, we also obtain a new formula for the tree-level S matrix of 4D N=4 Einstein-Maxwell theory.
RESUMO
PURPOSE: One response to the opioid crisis in the United States has been the development of opioid analgesics with properties intended to reduce non-oral use. Previous evaluations of abuse in the community have relied on population averaged interrupted time series Poisson models with utilization offsets. However, competing interventions and secular trends complicate interpretation of time-series analyses. An alternative research design, trend-in-trend, accounts for heterogeneity in per capita opioid dispensing and unmeasured time-varying confounding, which provides a causal evaluation, provided that underlying assumptions are met. METHODS: Trend-in-trend can be modeled using a logistic regression framework. In logistic regression, exposure was any product-specific outpatient dispensing by three-digit ZIP code and calendar quarter, for 22 opioids. The outcome was any product-specific abuse case ascertained from poison centers and drug treatment programs, covering 94% of the US population, between July 2009 and December 2016. Product-specific odds ratios compared places without dispensing with places with any dispensing; the causal contrast represents the odds of product-specific abuse in the community given exposure. RESULTS: Dispensing of new and low-volume opioids varied considerably across the country, with no region showing high of all products. Of 22 opioids analyzed, the three with approved labeling as intended to deter abuse ranked near the lowest in both absolute (population-adjusted rates: 1.7, 0.9, and 8.2 per million people per quarter, respectively) and relative measures (trend-in-trend ORs: 1.96, 1.79, 1.69, respectively). CONCLUSIONS: Postmarketing studies of prescription opioid abuse may benefit by evolving from unadjusted surveillance rates to a causal inference approach.
Assuntos
Analgésicos Opioides/administração & dosagem , Prescrições de Medicamentos/estatística & dados numéricos , Transtornos Relacionados ao Uso de Opioides/epidemiologia , Vigilância de Produtos Comercializados/estatística & dados numéricos , Humanos , Modelos Logísticos , Padrões de Prática Médica/estatística & dados numéricos , Estados UnidosRESUMO
Oysters from a reef in Galveston Bay, Texas, USA, were screened for more virulent clinical strains versus less virulent environmental strains of Vibrio vulnificus using a combination of quantitative PCR assays for the virulence correlating gene (clinical variant, vcgC) and 16S rRNA types A and B (type A = environmental, type B = clinical). The combination of vcgC and 16S rRNA type B loci to determine clinical type strains was suitable, as indicated by the strong correlation (R2 = 0.98; p < 0.001) between these gene counts over time and their relative proportion (up to 93.8% and 94.3%, respectively) to vvhA genes used to quantify all strains of V. vulnificus. A strong seasonal shift of V. vulnificus strain types was observed. Environmental strains (16S rRNA type A) predominated from April to mid-June as salinities increased from 22 to 27 PSU (practical salinity unit) and temperatures rose 20 to 28 °C, with peak gene quantities of 16 812 ± 56 CFU/g. As temperatures increased to ≥30 °C from mid-June to September and salinities rose above 27 PSU, clinical strains (16S rRNA type B; vcgC) predominated with peak quantities 31 868 ± 287 and 32 360 ± 178 CFU/g, respectively.
Assuntos
Proteínas de Bactérias/genética , Ostreidae/microbiologia , RNA Ribossômico 16S/genética , Vibrioses/microbiologia , Vibrio vulnificus/isolamento & purificação , Vibrio vulnificus/patogenicidade , Animais , Proteínas de Bactérias/metabolismo , Baías , DNA Bacteriano/genética , Contaminação de Alimentos/análise , Humanos , Reação em Cadeia da Polimerase/métodos , Estações do Ano , Vibrio vulnificus/classificação , Vibrio vulnificus/genética , Virulência/genéticaRESUMO
OBJECTIVE: Laminar flow activates myocyte enhancer factor 2 (MEF2) transcription factors in vitro to induce expression of atheroprotective genes in the endothelium. Here we sought to establish the role of Mef2c in the vascular endothelium in vivo. APPROACH AND RESULTS: To study endothelial Mef2c, we generated endothelial-specific deletion of Mef2c using Tie2-Cre or Cdh5-Cre-ERT2 and examined aortas and carotid arteries by en face immunofluorescence. We observed enhanced actin stress fiber formation in the Mef2c-deleted thoracic aortic endothelium (laminar flow region), similar to those observed in normal aortic inner curvature (disturbed flow region). Furthermore, Mef2c deletion resulted in the de novo formation of subendothelial intimal cells expressing markers of differentiated smooth muscle in the thoracic aortas and carotids. Lineage tracing showed that these cells were not of endothelial origin. To define early events in intimal development, we induced endothelial deletion of Mef2c and examined aortas at 4 and 12 weeks postinduction. The number of intimal cell clusters increased from 4 to 12 weeks, but the number of cells within a cluster peaked at 2 cells in both cases, suggesting ongoing migration but minimal proliferation. Moreover, we identified cells extending from the media through fenestrations in the internal elastic lamina into the intima, indicating transfenestral smooth muscle migration. Similar transfenestral migration was observed in wild-type carotid arteries ligated to induce neointimal formation. CONCLUSIONS: These results indicate that endothelial Mef2c regulates the endothelial actin cytoskeleton and inhibits smooth muscle cell migration into the intima.
Assuntos
Lesões das Artérias Carótidas/metabolismo , Movimento Celular , Células Endoteliais/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Comunicação Parácrina , Túnica Íntima/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Aorta Torácica/fisiopatologia , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Artérias Carótidas/fisiopatologia , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , Lesões das Artérias Carótidas/fisiopatologia , Linhagem da Célula , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/patologia , Genótipo , Hemodinâmica , Humanos , Fatores de Transcrição MEF2/deficiência , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Camundongos Knockout , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/patologia , Neointima , Fenótipo , Interferência de RNA , Fluxo Sanguíneo Regional , Transdução de Sinais , Fatores de Tempo , Transfecção , Túnica Íntima/patologia , Túnica Íntima/fisiopatologiaRESUMO
OBJECTIVE: The role of hemoglobin and myoglobin in the cardiovascular system is well established, yet other globins in this context are poorly characterized. Here, we examined the expression and function of cytoglobin (CYGB) during vascular injury. APPROACH AND RESULTS: We characterized CYGB content in intact vessels and primary vascular smooth muscle (VSM) cells and used 2 different vascular injury models to examine the functional significance of CYGB in vivo. We found that CYGB was strongly expressed in medial arterial VSM and human veins. In vitro and in vivo studies indicated that CYGB was lost after VSM cell dedifferentiation. In the rat balloon angioplasty model, site-targeted delivery of adenovirus encoding shRNA specific for CYGB prevented its reexpression and decreased neointima formation. Similarly, 4 weeks after complete ligation of the left common carotid, Cygb knockout mice displayed little to no evidence of neointimal hyperplasia in contrast to their wild-type littermates. Mechanistic studies in the rat indicated that this was primarily associated with increased medial cell loss, terminal uridine nick-end labeling staining, and caspase-3 activation, all indicative of prolonged apoptosis. In vitro, CYGB could be reexpressed after VSM stimulation with cytokines and hypoxia and loss of CYGB sensitized human and rat aortic VSM cells to apoptosis. This was reversed after antioxidant treatment or NOS2 (nitric oxide synthase 2) inhibition. CONCLUSIONS: These results indicate that CYGB is expressed in vessels primarily in differentiated medial VSM cells where it regulates neointima formation and inhibits apoptosis after injury.
Assuntos
Apoptose , Globinas/fisiologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiopatologia , Remodelação Vascular/fisiologia , Animais , Caspase 3/metabolismo , Diferenciação Celular , Citoglobina , Regulação para Baixo , Ativação Enzimática , Camundongos , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Neointima/fisiopatologia , Óxido Nítrico Sintase Tipo II/toxicidade , Oxirredução , RatosRESUMO
Numb family proteins (NFPs), including Numb and numb-like (Numbl), are cell fate determinants for multiple progenitor cell types. Their functions in cardiac progenitor differentiation and cardiac morphogenesis are unknown. To avoid early embryonic lethality and study NFP function in later cardiac development, Numb and Numbl were deleted specifically in heart to generate myocardial double-knockout (MDKO) mice. MDKOs were embryonic lethal and displayed a variety of defects in cardiac progenitor differentiation, cardiomyocyte proliferation, outflow tract (OFT) and atrioventricular septation, and OFT alignment. By ablating NFPs in different cardiac populations followed by lineage tracing, we determined that NFPs in the second heart field (SHF) are required for OFT and atrioventricular septation and OFT alignment. MDKOs displayed an SHF progenitor cell differentiation defect, as revealed by a variety of methods including mRNA deep sequencing. Numb regulated cardiac progenitor cell differentiation in an endocytosis-dependent manner. Studies including the use of a transgenic Notch reporter line showed that Notch signaling was upregulated in the MDKO. Suppression of Notch1 signaling in MDKOs rescued defects in p57 expression, proliferation and trabecular thickness. Further studies showed that Numb inhibits Notch1 signaling by promoting the degradation of the Notch1 intracellular domain in cardiomyocytes. This study reveals that NFPs regulate trabecular thickness by inhibiting Notch1 signaling, control cardiac morphogenesis in a Notch1-independent manner, and regulate cardiac progenitor cell differentiation in an endocytosis-dependent manner. The function of NFPs in cardiac progenitor differentiation and cardiac morphogenesis suggests that NFPs might be potential therapeutic candidates for cardiac regeneration and congenital heart diseases.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Coração/embriologia , Proteínas de Membrana/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem da Célula , Proliferação de Células , Feminino , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/metabolismo , Cardiopatias Congênitas/patologia , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Morfogênese/genética , Morfogênese/fisiologia , Miocárdio/citologia , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Gravidez , Receptor Notch1/genética , Receptor Notch1/metabolismo , Transdução de SinaisRESUMO
Vascular smooth muscle (VSM) expresses calcium/calmodulin-dependent protein kinase II (CaMKII)-δ and -γ isoforms. CaMKIIδ promotes VSM proliferation and vascular remodeling. We tested CaMKIIγ function in vascular remodeling after injury. CaMKIIγ protein decreased 90% 14 d after balloon injury in rat carotid artery. Intraluminal transduction of adenovirus encoding CaMKIIγC rescued expression to 35% of uninjured controls, inhibited neointima formation (>70%), inhibited VSM proliferation (>60%), and increased expression of the cell-cycle inhibitor p21 (>2-fold). Comparable doses of CaMKIIδ2 adenovirus had no effect. Similar dynamics in CaMKIIγ mRNA and protein expression were observed in ligated mouse carotid arteries, correlating closely with expression of VSM differentiation markers. Targeted deletion of CaMKIIγ in smooth muscle resulted in a 20-fold increase in neointimal area, with a 3-fold increase in the cell proliferation index, no change in apoptosis, and a 60% decrease in p21 expression. In cultured VSM, CaMKIIγ overexpression induced p53 mRNA (1.7 fold) and protein (1.8-fold) expression; induced the p53 target gene p21 (3-fold); decreased VSM cell proliferation (>50%); and had no effect on expression of apoptosis markers. We conclude that regulated CaMKII isoform composition is an important determinant of the injury-induced vasculoproliferative response and that CaMKIIγ and -δ isoforms have nonequivalent, opposing functions.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proliferação de Células/fisiologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Remodelação Vascular/fisiologia , Animais , Apoptose/fisiologia , Biomarcadores/metabolismo , Artérias Carótidas/metabolismo , Artérias Carótidas/fisiologia , Diferenciação Celular/fisiologia , Linhagem Celular , Masculino , Camundongos , Camundongos Knockout , Neointima/metabolismo , Neointima/patologia , Ratos , Ratos Sprague-DawleyRESUMO
RATIONALE: Our previous study has shown that yes-associated protein (YAP) plays a crucial role in the phenotypic modulation of vascular smooth muscle cells (SMCs) in response to arterial injury. However, the role of YAP in vascular SMC development is unknown. OBJECTIVE: The goal of this study was to investigate the functional role of YAP in cardiovascular development in mice and determine the mechanisms underlying YAP's actions. METHODS AND RESULTS: YAP was deleted in cardiomyocytes and vascular SMCs by crossing YAP flox mice with SM22α-Cre transgenic mice. Cardiac/SMC-specific deletion of YAP directed by SM22α-Cre resulted in perinatal lethality in mice because of profound cardiac defects including hypoplastic myocardium, membranous ventricular septal defect, and double outlet right ventricle. The cardiac/SMC-specific YAP knockout mice also displayed severe vascular abnormalities including hypoplastic arterial wall, short/absent brachiocephalic artery, and retroesophageal right subclavian artery. Deletion of YAP in mouse vascular SMCs induced expression of a subset of cell cycle arrest genes including G-protein-coupled receptor 132 (Gpr132). Silencing Gpr132 promoted SMC proliferation, whereas overexpression of Gpr132 attenuated SMC growth by arresting cell cycle in G0/G1 phase, suggesting that ablation of YAP-induced impairment of SMC proliferation was mediated, at least in part, by induction of Gpr132 expression. Mechanistically, YAP recruited the epigenetic repressor histone deacetylase-4 to suppress Gpr132 gene expression via a muscle CAT element in the Gpr132 gene. CONCLUSIONS: YAP plays a critical role in cardiac/SMC proliferation during cardiovascular development by epigenetically regulating expression of a set of cell cycle suppressors.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Anormalidades Cardiovasculares/genética , Coração Fetal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Miócitos Cardíacos/citologia , Miócitos de Músculo Liso/citologia , Fosfoproteínas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Aneurisma/genética , Animais , Tronco Braquiocefálico/anormalidades , Anormalidades Cardiovasculares/embriologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Divisão Celular , Células Cultivadas , Dupla Via de Saída do Ventrículo Direito/embriologia , Dupla Via de Saída do Ventrículo Direito/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Letais , Genes cdc , Comunicação Interventricular/embriologia , Comunicação Interventricular/genética , Histona Desacetilases/metabolismo , Histona Desacetilases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Músculo Liso Vascular/patologia , Miócitos Cardíacos/patologia , Miócitos de Músculo Liso/patologia , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Ratos , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/genética , Artéria Subclávia/anormalidades , Proteínas de Sinalização YAPRESUMO
Studies of determinants of development of inhibitory Abs to factor VIII in people with hemophilia A indicate a complex process involving multiple factors. The Hemophilia Inhibitor Genetics Study (HIGS) Combined Cohort was formed to extend our understanding of the genetic background of risk. The study group contains 833 subjects from 3 independent cohorts: brother pairs and singletons with and without a history of inhibitors, as well as 104 brother pairs discordant for inhibitor status. Using an Illumina iSelect platform, 13 331 single-nucleotide polymorphisms from 1081 genes, primarily immune response and immune modifier genes, were typed. Each cohort was analyzed separately with results combined using a meta-analytic technique. After adjustment for potential confounders, 53 single-nucleotide polymorphisms were found to be significant predictors of inhibitor status using the criteria of odds ratios in the same direction in all cohorts or allowing for a 20% interval around an odds ratio = 1 in 1 of the 3 and significant in at least 2. Of the 53 markers, 13 had meta P < .001. Eight of the 53 were significant predictors among the discordant pairs. Results support the complexity of the immune response and encourage further research with the goal of understanding the pathways involved.
Assuntos
Fator VIII/imunologia , Fator VIII/uso terapêutico , Hemofilia A , Herança Multifatorial/genética , Transcriptoma , Adolescente , Anticorpos/imunologia , Criança , Estudos de Coortes , Resistência a Medicamentos/genética , Resistência a Medicamentos/imunologia , Fator VIII/genética , Marcadores Genéticos , Predisposição Genética para Doença/epidemiologia , Predisposição Genética para Doença/genética , Hemofilia A/tratamento farmacológico , Hemofilia A/epidemiologia , Hemofilia A/genética , Humanos , Masculino , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco , IrmãosAssuntos
Histona Desacetilases/farmacologia , Hipertensão Pulmonar/terapia , Fatores de Transcrição MEF2/farmacologia , Animais , Modelos Animais de Doenças , Imunofluorescência , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Hipertensão Pulmonar/genética , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , CamundongosRESUMO
BACKGROUND: DNA methylation integrates environmental signals with transcriptional programs. COVID-19 infection induces changes in the host methylome. While post-acute sequelae of COVID-19 (PASC) is a long-term complication of acute illness, its association with DNA methylation is unknown. No universal blood marker of PASC, superseding single organ dysfunctions, has yet been identified. METHODS: In this single centre prospective cohort study, PASC, post-COVID without PASC, and healthy participants were enrolled to investigate their symptoms association with peripheral blood DNA methylation data generated with state-of-the-art whole genome sequencing. PASC-induced quality-of-life deterioration was scored with a validated instrument, SF-36. Analyses were conducted to identify potential functional roles of differentially methylated loci, and machine learning algorithms were used to resolve PASC severity. FINDINGS: 103 patients with PASC (22.3% male, 77.7% female), 15 patients with previous COVID-19 infection but no PASC (40.0% male, 60.0% female), and 27 healthy volunteers (48.1% male, 51.9% female) were enrolled. Whole genome methylation sequencing revealed 39 differentially methylated regions (DMRs) specific to PASC, each harbouring an average of 15 consecutive positions, that differentiate patients with PASC from the two control groups. Motif analyses of PASC-regulated DMRs identify binding domains for transcription factors regulating circadian rhythm and others. Some DMRs annotated to protein coding genes were associated with changes of RNA expression. Machine learning support vector algorithm and random forest hierarchical clustering reveal 28 unique differentially methylated positions (DMPs) in the genome discriminating patients with better and worse quality of life. INTERPRETATION: Blood DNA methylation levels identify PASC, stratify PASC severity, and suggest that DNA motifs are targeted by circadian rhythm-regulating pathways in PASC. FUNDING: This project has been funded by the following agencies: NIH-AI173035 (A. Jaitovich and R. Alisch); and NIH-AG066179 (R. Alisch).
RESUMO
Ischemic retinopathies, including retinopathy of prematurity and diabetic retinopathy, are major causes of blindness. Both have two phases, vessel loss and consequent hypoxia-driven pathologic retinal neovascularization, yet relatively little is known about the transcription factors regulating these processes. Myocyte enhancer factor 2 (MEF2) C, a member of the MEF2 family of transcription factors that plays an important role in multiple developmental programs, including the cardiovascular system, seems to have a significant functional role in the vasculature. We, therefore, generated endothelial cell (EC)-specific MEF2C-deficient mice and explored the role of MEF2C in retinal vascularization during normal development and in a mouse model of oxygen-induced retinopathy. Ablation of MEF2C did not cause appreciable defects in normal retinal vascular development. However, MEF2C ablation in ECs suppressed vessel loss in oxygen-induced retinopathy and strongly promoted vascular regrowth, consequently reducing retinal avascularity. This finding was associated with suppression of pathologic retinal angiogenesis and blood-retinal barrier dysfunction. MEF2C knockdown in cultured retinal ECs using small-interfering RNAs rescued ECs from death and stimulated tube formation under stress conditions, confirming the endothelial-autonomous and antiangiogenic roles of MEF2C. HO-1 was induced by MEF2C knockdown in vitro and may play a role in the proangiogenic effect of MEF2C knockdown on retinal EC tube formation. Thus, MEF2C may play an antiangiogenic role in retinal ECs under stress conditions, and modulation of MEF2C may prevent pathologic retinal neovascularization.
Assuntos
Fatores de Regulação Miogênica/fisiologia , Neovascularização Retiniana/fisiopatologia , Vasos Retinianos/patologia , Retinopatia da Prematuridade/fisiopatologia , Animais , Apoptose/fisiologia , Barreira Hematorretiniana/fisiologia , Células Cultivadas , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Técnicas de Silenciamento de Genes , Humanos , Recém-Nascido , Fatores de Transcrição MEF2 , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fatores de Regulação Miogênica/genética , Fatores de Regulação Miogênica/metabolismo , Estresse Oxidativo/fisiologia , Oxigênio , RNA Interferente Pequeno/genética , Neovascularização Retiniana/genética , Neovascularização Retiniana/prevenção & controle , Vasos Retinianos/crescimento & desenvolvimento , Vasos Retinianos/metabolismo , Retinopatia da Prematuridade/patologiaRESUMO
VSMCs (vascular smooth muscle cells) dedifferentiate from the contractile to the synthetic phenotype in response to acute vascular diseases such as restenosis and chronic vascular diseases such as atherosclerosis, and contribute to growth of the neointima. We demonstrated previously that balloon catheter injury of rat carotid arteries resulted in increased expression of CaMKII (Ca(2+)/calmodulin-dependent protein kinase) IIδ(2) in the medial wall and the expanding neointima [House and Singer (2008) Arterioscler. Thromb. Vasc. Biol. 28, 441-447]. These findings led us to hypothesize that increased expression of CaMKIIδ(2) is a positive mediator of synthetic VSMCs. HDAC (histone deacetylase) 4 and HDAC5 function as transcriptional co-repressors and are regulated in a CaMKII-dependent manner. In the present paper, we report that endogenous HDAC4 and HDAC5 in VSMCs are activated in a Ca(2+)- and CaMKIIδ(2)-dependent manner. We show further that AngII (angiotensin II)- and PDGF (platelet-derived growth factor)-dependent phosphorylation of HDAC4 and HDAC5 is reduced when CaMKIIδ(2) expression is suppressed or CaMKIIδ(2) activity is attenuated. The transcriptional activator MEF2 (myocyte-enhancer factor 2) is an important determinant of VSMC phenotype and is regulated in an HDAC-dependent manner. In the present paper, we report that stimulation of VSMCs with ionomycin or AngII potentiates MEF2's ability to bind DNA and increases the expression of established MEF2 target genes Nur77 (nuclear receptor 77) (NR4A1) and MCP1 (monocyte chemotactic protein 1) (CCL2). Suppression of CaMKIIδ(2) attenuates increased MEF2 DNA-binding activity and up-regulation of Nur77 and MCP1. Finally, we show that HDAC5 is regulated by HDAC4 in VSMCs. Suppression of HDAC4 expression and activity prevents AngII- and PDGF-dependent phosphorylation of HDAC5. Taken together, these results illustrate a mechanism by which CaMKIIδ(2) mediates MEF2-dependent gene transcription in VSMCs through regulation of HDAC4 and HDAC5.
Assuntos
Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/fisiologia , Histona Desacetilases/fisiologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fatores de Regulação Miogênica/metabolismo , Angiotensina II/farmacologia , Animais , Aorta Torácica/citologia , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Isoenzimas/fisiologia , Fatores de Transcrição MEF2 , Músculo Liso Vascular/citologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Multimerização Proteica , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Transcrição GênicaRESUMO
Sox17 is a critical regulator of arterial identity during early embryonic vascular development. However, its role in adult endothelial cells (ECs) are not fully understood. Sox17 is highly expressed in arterial ECs but not in venous ECs throughout embryonic development to adulthood suggesting that it may play a functional role in adult arteries. Here, we investigated Sox17 mediated phenotypical changes in adult ECs. To precisely control the temporal expression level of Sox17, we designed a tetracycline-inducible lentiviral gene expression system to express Sox17 selectively in cultured venous ECs. We confirmed that Sox17-induced ECs exhibit a gene profile favoring arterial and tip cell identity. Furthermore, in comparison to control ECs, Sox17-activated ECs under shear leads to greater expression of arterial markers and suppression of venous identity. These data suggest that Sox17 enables greater hemodynamic adaptability of ECs in response to fluid shear stress. Here, we also demonstrate key morphogenic behaviors of Sox17-mediated ECs. In both vasculogenic and angiogenic 3D fibrin gel studies, Sox17-mediated ECs prefer to form cohesive vessels with one another while interfering the vessel formation of the control ECs. Sox17-mediated ECs elicit hyper-sprouting behavior in the presence of pericytes but not fibroblasts, suggesting Sox17 mediated sprouting frequency is dependent on supporting cell type. Using a microfluidic chip, we also show that Sox17-mediated ECs maintain thinner diameter vessels that do not widen under interstitial flow like the control ECs. Taken together, these data showed that Sox17 mediated EC gene expression and phenotypical changes are highly modulated in the context of biomechanical stimuli, suggesting Sox17 plays a role in regulating the arterial ECs adaptability under arterial hemodynamics as well as tip cells behavior during angiogenesis and vasculogenesis. The results from this study may be valuable in improving vein graft adaptation to arterial hemodynamics and bioengineering microvasculature for tissue engineering applications.