RESUMO
MicroRNAs are key regulators of angiogenesis, as illustrated by the vascular defects observed in miR-126-deficient animals. The miR-126 duplex gives rise to two mature microRNAs (miR-126-3p and -5p). The vascular defects in these mutant animals were attributed to the loss of miR-126-3p but the role of miR-126-5p during normal angiogenesis in vivo remains unknown. Here, we show that miR-126-5p is expressed in endothelial cells but also by retinal ganglion cells (RGCs) of the mouse postnatal retina and participates in protecting endothelial cells from apoptosis during the establishment of the retinal vasculature. miR-126-5p negatively controls class 3 semaphorin protein (Sema3A) in RGCs through the repression of SetD5, an uncharacterized member of the methyltransferase family of proteins. In vitro, SetD5 controls Sema3A expression independently of its SET domain and co-immunoprecipitates with BRD2, a bromodomain protein that recruits transcription regulators onto the chromatin. Both SetD5 and BRD2 bind to the transcription start site and to upstream promoter regions of the Sema3a locus and BRD2 is necessary for the regulation of Sema3A expression by SetD5. Thus, neuronally expressed miR-126-5p regulates angiogenesis by protecting endothelial cells of the developing retinal vasculature from apoptosis.
Assuntos
Apoptose/fisiologia , Células Endoteliais/metabolismo , Metiltransferases/biossíntese , MicroRNAs/biossíntese , Neurônios/metabolismo , Retina/metabolismo , Animais , Sobrevivência Celular/fisiologia , Células Endoteliais/citologia , Camundongos , Camundongos Knockout , MicroRNAs/genética , Neovascularização Fisiológica/fisiologia , Neurônios/citologia , Elementos de Resposta/fisiologia , Retina/citologia , Semaforina-3A/genética , Semaforina-3A/metabolismoRESUMO
Activation of the blood vessel endothelium is a critical step during inflammation. Endothelial cells stimulated by pro-inflammatory cytokines play an essential part in the adhesion and extravasation of circulating leukocytes into inflamed tissues. The endothelial egfl7 gene (VE-statin) represses endothelial cell activation in tumors, and prior observations suggested that it could also participate in the regulation of endothelial cell activation during inflammation. We show here that Egfl7 expression is strongly repressed in mouse lung endothelial cells during LPS- and TNFα-induced inflammation in vivo LPS have a limited effect on Egfl7 expression by endothelial cells in vitro, whereas the pro-inflammatory cytokine TNFα strongly represses Egfl7 expression in endothelial cells. TNFα regulates the egfl7 gene promoter through regions located between -7585 and -5550 bp ahead of the main transcription start site and via an NF-κB-dependent mechanism. Conversely, Egfl7 regulates the response of endothelial cells to TNFα by restraining the induced expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, resulting in a decreased adhesion of leukocytes onto endothelial cells stimulated by TNFα. Egfl7 regulates the expression of these adhesion molecules through the NF-κB and MEK/Erk pathways, in particular by preventing the proteasome-mediated degradation of IkBα both in non-activated endothelial cells and during activation. Egfl7 is thus an endogenous and constitutive repressor of blood vessel endothelial cell activation in normal and inflammatory conditions and participates in a loop of regulation of activation of these cells by pro-inflammatory cytokines.
Assuntos
Fatores de Crescimento Endotelial/biossíntese , Regulação da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Elementos de Resposta , Animais , Proteínas de Ligação ao Cálcio , Família de Proteínas EGF , Fatores de Crescimento Endotelial/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Células Jurkat , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Photosynthetic algae have evolved mechanisms to cope with suboptimal light and CO2 conditions. When light energy exceeds CO2 fixation capacity, Chlamydomonas reinhardtii activates photoprotection, mediated by LHCSR1/3 and PSBS, and the CO2 Concentrating Mechanism (CCM). How light and CO2 signals converge to regulate these processes remains unclear. Here, we show that excess light activates photoprotection- and CCM-related genes by altering intracellular CO2 concentrations and that depletion of CO2 drives these responses, even in total darkness. High CO2 levels, derived from respiration or impaired photosynthetic fixation, repress LHCSR3/CCM genes while stabilizing the LHCSR1 protein. Finally, we show that the CCM regulator CIA5 also regulates photoprotection, controlling LHCSR3 and PSBS transcript accumulation while inhibiting LHCSR1 protein accumulation. This work has allowed us to dissect the effect of CO2 and light on CCM and photoprotection, demonstrating that light often indirectly affects these processes by impacting intracellular CO2 levels.
Assuntos
Dióxido de Carbono , Chlamydomonas reinhardtii , Dióxido de Carbono/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Fotossíntese/genética , Proteínas/metabolismo , Chlamydomonas reinhardtii/metabolismoRESUMO
Sanguina nivaloides is the main alga forming red snowfields in high mountains and Polar Regions. It is non-cultivable. Analysis of environmental samples by X-ray tomography, focused-ion-beam scanning-electron-microscopy, physicochemical and physiological characterization reveal adaptive traits accounting for algal capacity to reside in snow. Cysts populate liquid water at the periphery of ice, are photosynthetically active, can survive for months, and are sensitive to freezing. They harbor a wrinkled plasma membrane expanding the interface with environment. Ionomic analysis supports a cell efflux of K+, and assimilation of phosphorus. Glycerolipidomic analysis confirms a phosphate limitation. The chloroplast contains thylakoids oriented in all directions, fixes carbon in a central pyrenoid and produces starch in peripheral protuberances. Analysis of cells kept in the dark shows that starch is a short-term carbon storage. The biogenesis of cytosolic droplets shows that they are loaded with triacylglycerol and carotenoids for long-term carbon storage and protection against oxidative stress.
Assuntos
Cistos , Neve , Humanos , Cloroplastos/metabolismo , Cistos/metabolismo , Carbono/metabolismo , Amido/metabolismoRESUMO
The tumor blood vessel endothelium forms a barrier that must be crossed by circulating immune cells in order for them to reach and kill cancer cells. Epidermal growth factorlike domain 7 (Egfl7) represses this immune infiltration by lowering the expression levels of leukocyte adhesion receptors on the surface of endothelial cells. However, the protein domains involved in these properties are not completely understood. Egfl7 is structurally composed of the predicted EMI, EGF and Cterminal domains. The present study aimed to investigate the roles of these different domains in tumor development by designing retroviruses coding for deletion mutants and then infecting 4T1 breast cancer cell populations, which consequently overexpressed the variants. By performing in vitro softagar assays, it was found that Egfl7 and its deletion variants did not affect cell proliferation or anchorageindependent growth. When 4T1 cells expressing either the wildtype Egfl7 protein or Egfl7 domain variants were implanted in mice, Egfl7 expression markedly promoted tumor development and deletion of the EGF repeats decreased the tumor growth rate. By contrast, deleting any other domain displayed no significant effect on tumor development. The overexpression of Egfl7 also decreased T cell and natural killer cell infiltration in tumors, as determined by immunofluorescence staining of tumor sections, whereas deletion of the EGF repeats inhibited this effect. Reverse transcriptionquantitative PCR analysis of the mechanisms involved revealed that deleting the EGF repeats partially restored the expression levels of vascular cell adhesion molecule 1 and Eselectin, which were suppressed by overexpression of Egfl7 in endothelial cells in vitro. This resulted in a higher number of lymphocytes bound to HUVEC expressing Egfl7ΔEGF compared with HUVEC expressing wildtype Egfl7, as assessed by fluorescentTHP1 adhesion assays onto endothelial cells. Overall, the present study demonstrated that the EGF repeats may participate in the protumoral and antiinflammatory effects of Egfl7.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Família de Proteínas EGF/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Sistema Imunitário/metabolismo , Evasão Tumoral/genética , Animais , Proteínas de Ligação ao Cálcio/genética , Proliferação de Células/genética , Modelos Animais de Doenças , Regulação para Baixo , Família de Proteínas EGF/genética , Feminino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
In nature, photosynthetic organisms are exposed to different light spectra and intensities depending on the time of day and atmospheric and environmental conditions. When photosynthetic cells absorb excess light, they induce nonphotochemical quenching to avoid photodamage and trigger expression of "photoprotective" genes. In this work, we used the green alga Chlamydomonas reinhardtii to assess the impact of light intensity, light quality, photosynthetic electron transport, and carbon dioxide on induction of the photoprotective genes (LHCSR1, LHCSR3, and PSBS) during dark-to-light transitions. Induction (mRNA accumulation) occurred at very low light intensity and was independently modulated by blue and ultraviolet B radiation through specific photoreceptors; only LHCSR3 was strongly controlled by carbon dioxide levels through a putative enhancer function of CIA5, a transcription factor that controls genes of the carbon concentrating mechanism. We propose a model that integrates inputs of independent signaling pathways and how they may help the cells anticipate diel conditions and survive in a dynamic light environment.
RESUMO
Mice deficient in the runt homology domain transcription factor Runx1/AML1 fail to generate functional clonogenic hematopoietic cells and die in utero by embryonic day 12.5. We previously generated Runx1 reversible knockout mice, in which the Runx1 locus can be restored by Cre-mediated recombination. We show here that selective restoration of the Runx1 locus in the Tie2 cell compartment rescues clonogenic hematopoietic progenitors in early Runx1-null embryos and rescues lymphoid and myeloid lineages during fetal development. Furthermore, fetal liver cells isolated from reactivated Runx1 embryos are capable of long-term multilineage lymphomyeloid reconstitution of adult irradiated recipients, demonstrating the rescue of definitive hematopoietic stem cells. However, this rescue of the definitive hematopoietic hierarchy is not sufficient to rescue the viability of animals beyond birth, pointing to an essential role for Runx1 in other vital developmental processes.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Animais , Southern Blotting , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Feminino , Imuno-Histoquímica , Integrases/genética , Integrases/fisiologia , Masculino , Camundongos , Camundongos Knockout , Modelos Teóricos , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/fisiologia , Receptor TIE-2RESUMO
The extracellular matrix (ECM) is essential to provide mechanical support to tissues but is also a bioactive edifice which controls cell behavior. Cell signaling generated by ECM components through integrin-mediated contacts, modulates cell biological activity. In addition, by sequestrating or releasing growth factors, the ECM is an active player of physiological and pathological processes such as vascular development. EGFL7 is mainly expressed during blood vessel development and is deposited in the ECM after secretion by endothelial cells. While EGFL7 is known to control various endothelial cell molecular mechanisms [i.e., the repression of endothelial-derived lysyl oxidase (LOX) enzyme, the regulation of the Notch pathway, and the expression of leukocyte adhesion molecules and of RHOA by endothelial cells], it is not established whether EGFL7 functions when bound to the ECM. Here, we show that microfibrillar-associated glycoprotein-1 (MAGP-1) and fibronectin drive the deposition of EGFL7 into both fibers and individual aggregates in endothelial ECM. Although EGFL7 does not need to be docked into the ECM to control endothelial adhesion molecule expression, the ECM accumulation of EGFL7 is required for its regulation of LOX activity and of HEY2 expression along the Notch pathway. The interaction of EGFL7 with MAGP-1 is necessary for LOX activity repression by EGFL7 while it does not participate in the control of the Notch pathway by this protein. Altogether, this study highlights the roles played by EGFL7 in controlling various endothelial molecular mechanisms upon its localization and shows how the ECM can modulate its functions.
Assuntos
Adesão Celular , Proteínas Contráteis/metabolismo , Fatores de Crescimento Endotelial/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Transdução de Sinais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação ao Cálcio , Família de Proteínas EGF , Humanos , Proteína-Lisina 6-Oxidase/metabolismo , Fatores de Processamento de RNA , Receptores Notch/metabolismo , Proteínas Repressoras/metabolismoRESUMO
A sensitive and accurate stable isotope dilution GC/MS assay was developed and validated for the quantification of succinylacetone (SA) in plasma and amniotic fluid (AF). SA is pathognonomic for tyrosinemia type I, a genetic disorder caused by a reduced activity of fumarylacetoacetate hydrolase (FAH). In untreated patients, SA can easily be measured in plasma and urine because the expected concentrations are in the micromol/L range. Due to a founder effect, the province of Quebec has an unusually high prevalence of tyrosinemia type I, hence, the quantification of SA in AF or plasma of treated patients in the nmol/L range becomes very useful. The method utilizes 13C5-SA as an internal standard and a three-step sample treatment consisting of oximation, solvent extraction and TMCS derivatization. The assay was validated by recording the ion intensities of m/z 620 for SA and m/z 625 for ISTD in order to demonstrate the precision of measurements, the linearity of the method, limit of quantification and detection (LOQ and LOD), specificity, accuracy, as well as metabolite stability. Values for the intra-day assays ranged from 0.2 to 3.2% while values for the inter-day assays ranged from 1.9 to 5.6% confirming that the method has good precision. A calibration plot using SA detected by GC/MS gave excellent linearity with a correlation coefficient of 0.999 over the injected concentration range of 5-2000 nmol/L. LOQ and LOD were 3 and 1 nmol/L, respectively. The usefulness of this method was demonstrated by SA quantification in an AF sample of an affected fetus and in plasma of patients treated with NTBC. The results demonstrate that this novel GC/MS method may be a valuable tool for metabolic evaluation and clinical use.
Assuntos
Líquido Amniótico/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Heptanoatos/análise , Tirosinemias/diagnóstico , Heptanoatos/sangue , Humanos , Monitorização Fisiológica , Nanotecnologia , Reprodutibilidade dos Testes , Tirosinemias/sangue , Tirosinemias/fisiopatologiaRESUMO
Tumor blood vessels participate in the immune response against cancer cells and we previously used pre-clinical models to demonstrate that egfl7 (VE-statin) promotes tumor cell evasion from the immune system by repressing endothelial cell activation, preventing immune cells from entering the tumor mass. In the present study, the expression levels of egfl7 and that of ICAM-1 as a marker of endothelium activation, were evaluated in peritumoral vessels of human breast cancer samples. Breast cancer samples (174 invasive and 30 in situ) from 204 patients treated in 2005 were immunostained for CD31, ICAM-1 and stained for egfl7 using in situ hybridization. The expression levels of ICAM-1 and egfl7 were assessed in peritumoral areas using semi-quantitative scales. There was a strong and significant inverse correlation between the expression of ICAM-1 and that of egfl7 in CD31+ blood vessels. When the ICAM-1 score increased, the egfl7 score reduced significantly (P=0.004), and vice-versa (Cuzick's test for trend across ordered groups). In order to determine which gene influenced the other gene between egfl7 and ICAM-1, the expression levels of either gene were modulated in endothelial cells. Egfl7 regulated ICAM-1 expression while ICAM-1 had no effects on egfl7 expression in the same conditions. Altogether, these results provide further results that egfl7 serves a regulatory role in endothelial cell activation in relation to immune infiltration and that it is a potential therapeutic target to consider for improving anticancer immunotherapies.
RESUMO
AIMS: miR126-5p is processed from the miR126-3p/-5p duplex, which is expressed in endothelial cells and gives rise to the guide strand miR126-3p and the passenger strand miR126-5p. miR126-3p has prominent roles in vascular development and diseases, whereas the expression and physiological functions of miR126-5p are unknown. The purpose of this study was to evaluate the expression and role of miR126-5p in blood vessel endothelial cells. METHODS AND RESULTS: miR126-5p is mostly expressed in blood vessel endothelial cells in vivo and in vitro. Gain- and loss-of-function approaches revealed that miR126-5p promotes leucocyte adhesion and represses leucocyte transendothelial migration. Two distinct target genes of miR126-5p in endothelial cells were identified: the activated leucocyte cell adhesion molecule (ALCAM) gene which codes for an adhesion molecule involved in leucocyte transendothelial migration and SetD5, a gene with previously unknown functions. Using either a blocking antibody or target protectors which specifically disrupt the miRNA/mRNA target pairing, we showed that miR126-5p promotes leucocyte adhesion by controlling the expression of SetD5 and represses transendothelial migration via the regulation of ALCAM. miR126-5p controls ALCAM and SetD5 expression in vivo in separate tissues and regulates leucocyte infiltration into inflamed lungs by repressing ALCAM expression. CONCLUSION: miR126-5p is a functional, endothelial-enriched microRNA that participates in the control of leucocyte trafficking by regulating the expression of ALCAM and SetD5.
Assuntos
Antígenos CD/genética , Moléculas de Adesão Celular Neuronais/genética , Movimento Celular , Células Endoteliais/fisiologia , Proteínas Fetais/genética , Leucócitos/fisiologia , Metiltransferases/genética , MicroRNAs/fisiologia , Animais , Adesão Celular , Células Cultivadas , Regulação da Expressão Gênica , Humanos , CamundongosRESUMO
The vasculature of the central nervous system (CNS) is composed of vascular endothelial and mural cells which interact closely with glial cells and neurons. The development of the CNS vascularisation is a unique process which requires the contribution of specific regulators in addition to the classical angiogenic factors. The egfl7 gene is mainly detected in endothelial cells during physiological and pathological angiogenesis. Egfl7 codes for a secreted protein which predominantly accumulates into the extracellular space where it controls vascular elastin deposition or the Notch pathway. Egfl7 is the host gene of the microRNA miR126 which is also expressed in endothelial cells and which plays major functions during blood vessel development. While the expression of egfl7 and that of miR126 were well described in endothelial cells during development, their pattern of expression during the establishment of the CNS vasculature is still unknown. By analysing the expression of egfl7 and miR126 during mouse retina vascularisation, we observed that while expression of miR126 is detected in all endothelia, egfl7 is initially expressed in all endothelial cells and then is progressively restricted to veins and to their neighbouring capillaries. The recruitment of mural cells around retina arteries coincides with the down-regulation of egfl7 in the arterial endothelial cells, suggesting that this recruitment could be involved in the loss of egfl7 expression in arteries. However, the expression pattern of egfl7 is similar when mural cell recruitment is prevented by the injection of a PDGFRß blocking antibody, suggesting that vessel maturation is not responsible for egfl7 down-regulation in retinal arteries.
Assuntos
Fatores de Crescimento Endotelial/genética , Proteínas/genética , Artéria Retiniana/crescimento & desenvolvimento , Artéria Retiniana/metabolismo , Veia Retiniana/crescimento & desenvolvimento , Veia Retiniana/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Família de Proteínas EGF , Fatores de Crescimento Endotelial/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas/metabolismoRESUMO
Hematopoietic stem cells (HSCs) are produced by a small cohort of hemogenic endothelial cells (ECs) during development through the formation of intra-aortic hematopoietic cell (HC) clusters. The Runx1 transcription factor plays a key role in the EC-to-HC and -HSC transition. We show that Runx1 expression in hemogenic ECs and the subsequent initiation of HC formation are tightly controlled by the subaortic mesenchyme, although the mesenchyme is not a source of HCs. Runx1 and Notch signaling are involved in this process, with Notch signaling decreasing with time in HCs. Inhibiting Notch signaling readily increases HC production in mouse and chicken embryos. In the mouse, however, this increase is transient. Collectively, we show complementary roles of hemogenic ECs and mesenchymal compartments in triggering aortic hematopoiesis. The subaortic mesenchyme induces Runx1 expression in hemogenic-primed ECs and collaborates with Notch dynamics to control aortic hematopoiesis.
Assuntos
Aorta/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Células Endoteliais/metabolismo , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Animais , Aorta/crescimento & desenvolvimento , Proteínas de Ligação ao Cálcio , Diferenciação Celular/genética , Movimento Celular , Células Cultivadas , Galinhas , Subunidade alfa 2 de Fator de Ligação ao Core/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Hemangioblastos , Peptídeos e Proteínas de Sinalização Intercelular , Proteína Jagged-2 , Proteínas de Membrana , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Codorniz , Receptores Notch/metabolismo , Proteínas Serrate-Jagged , Transdução de Sinais/genéticaRESUMO
Downregulating the leukocyte adhesion molecules expressed by endothelial cells that line tumor blood vessels can limit the entry of immune effector cells into the tumor mass, thereby contributing to tumoral immune escape. Egfl7 (also known as VE-statin) is a secreted protein specifically expressed by endothelial cells in normal tissues and by cancer cells in various human tumors. High levels of Egfl7 correlate with higher tumor grade and poorer prognosis. Here we show that expression of Egfl7 in breast and lung carcinoma cells accelerates tumor growth and metastasis in immunocompetent mice but not in immunodeficient mice. Tumors expressing Egfl7 were infiltrated relatively poorly by immune cells and were characterized by reduced levels of immunostimulatory cytokines [IFN-γ, interleukin-12 (IL-12)] and fewer endothelial adhesion molecules [intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1)]. In vitro studies revealed that Egfl7 inhibited the expression of leukocyte adhesion molecules by endothelial cells, preventing lymphocyte adhesion. In contrast, Egfl7 did not exert any effects on immune cell activation. Human breast cancer lesions expressing high levels of Egfl7 also expressed less ICAM-1 and VCAM-1 in their blood vessels, also indicating an inverse correlation between expression levels of Egfl7 and IFN-γ. Thus, Egfl7 expression in tumors promotes tumor progression by reducing the expression of endothelial molecules that mediate immune cell infiltration. Our findings highlight a novel mechanism through which tumors escape immune control.