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1.
Immunity ; 55(12): 2285-2299.e7, 2022 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-36272416

RESUMEN

Intravascular neutrophils and platelets collaborate in maintaining host integrity, but their interaction can also trigger thrombotic complications. We report here that cooperation between neutrophil and platelet lineages extends to the earliest stages of platelet formation by megakaryocytes in the bone marrow. Using intravital microscopy, we show that neutrophils "plucked" intravascular megakaryocyte extensions, termed proplatelets, to control platelet production. Following CXCR4-CXCL12-dependent migration towards perisinusoidal megakaryocytes, plucking neutrophils actively pulled on proplatelets and triggered myosin light chain and extracellular-signal-regulated kinase activation through reactive oxygen species. By these mechanisms, neutrophils accelerate proplatelet growth and facilitate continuous release of platelets in steady state. Following myocardial infarction, plucking neutrophils drove excessive release of young, reticulated platelets and boosted the risk of recurrent ischemia. Ablation of neutrophil plucking normalized thrombopoiesis and reduced recurrent thrombosis after myocardial infarction and thrombus burden in venous thrombosis. We establish neutrophil plucking as a target to reduce thromboischemic events.


Asunto(s)
Enfermedades Cardiovasculares , Infarto del Miocardio , Trombosis , Humanos , Megacariocitos , Trombopoyesis , Neutrófilos , Plaquetas/fisiología
2.
Blood ; 139(8): 1184-1197, 2022 02 24.
Artículo en Inglés | MEDLINE | ID: mdl-33908607

RESUMEN

Cancer cells are in most instances characterized by rapid proliferation and uncontrolled cell division. Hence, they must adapt to proliferation-induced metabolic stress through intrinsic or acquired antimetabolic stress responses to maintain homeostasis and survival. One mechanism to achieve this is reprogramming gene expression in a metabolism-dependent manner. MondoA (also known as Myc-associated factor X-like protein X-interacting protein [MLXIP]), a member of the MYC interactome, has been described as an example of such a metabolic sensor. However, the role of MondoA in malignancy is not fully understood and the underlying mechanism in metabolic responses remains elusive. By assessing patient data sets, we found that MondoA overexpression is associated with worse survival in pediatric common acute lymphoblastic leukemia (ALL; B-precursor ALL [B-ALL]). Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and RNA-interference approaches, we observed that MondoA depletion reduces the transformational capacity of B-ALL cells in vitro and dramatically inhibits malignant potential in an in vivo mouse model. Interestingly, reduced expression of MondoA in patient data sets correlated with enrichment in metabolic pathways. The loss of MondoA correlated with increased tricarboxylic acid cycle activity. Mechanistically, MondoA senses metabolic stress in B-ALL cells by restricting oxidative phosphorylation through reduced pyruvate dehydrogenase activity. Glutamine starvation conditions greatly enhance this effect and highlight the inability to mitigate metabolic stress upon loss of MondoA in B-ALL. Our findings give novel insight into the function of MondoA in pediatric B-ALL and support the notion that MondoA inhibition in this entity offers a therapeutic opportunity and should be further explored.


Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Estrés Fisiológico , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Línea Celular Tumoral , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética
3.
Proc Natl Acad Sci U S A ; 116(27): 13414-13423, 2019 07 02.
Artículo en Inglés | MEDLINE | ID: mdl-31196952

RESUMEN

The molecular mechanisms regulating sympathetic innervation of the heart during embryogenesis and its importance for cardiac development and function remain to be fully elucidated. We generated mice in which conditional knockout (CKO) of the Hif1a gene encoding the transcription factor hypoxia-inducible factor 1α (HIF-1α) is mediated by an Islet1-Cre transgene expressed in the cardiac outflow tract, right ventricle and atrium, pharyngeal mesoderm, peripheral neurons, and hindlimbs. These Hif1aCKO mice demonstrate significantly decreased perinatal survival and impaired left ventricular function. The absence of HIF-1α impaired the survival and proliferation of preganglionic and postganglionic neurons of the sympathetic system, respectively. These defects resulted in hypoplasia of the sympathetic ganglion chain and decreased sympathetic innervation of the Hif1aCKO heart, which was associated with decreased cardiac contractility. The number of chromaffin cells in the adrenal medulla was also decreased, indicating a broad dependence on HIF-1α for development of the sympathetic nervous system.


Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Sistema Nervioso Simpático/crecimiento & desarrollo , Médula Suprarrenal/embriología , Médula Suprarrenal/inervación , Animales , Células Cromafines , Anomalías de los Vasos Coronarios/embriología , Vasos Coronarios/embriología , Femenino , Ganglios Simpáticos/embriología , Ganglios Simpáticos/crecimiento & desarrollo , Corazón/embriología , Corazón/inervación , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Sistema Nervioso Simpático/enzimología
4.
J Virol ; 89(17): 9080-9, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26109726

RESUMEN

UNLABELLED: Human T-cell leukemia virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and transforms T cells in vitro. To our knowledge, the functional role of reactive oxygen species (ROS)-generating NADPH oxidase 5 (Nox5) in HTLV-1 transformation remains undefined. Here, we found that Nox5α expression was upregulated in 88% of 17 ATL patient samples but not in normal peripheral blood T cells. Upregulation of the Nox5α variant was transcriptionally sustained by the constitutive Janus family tyrosine kinase (Jak)-STAT5 signaling pathway in interleukin-2 (IL-2)-independent HTLV-1-transformed cell lines, including MT1 and MT2, whereas it was transiently induced by the IL-2-triggered Jak-STAT5 axis in uninfected T cells. A Nox inhibitor, diphenylene iodonium, and antioxidants such as N-acetyl cysteine blocked proliferation of MT1 and MT2 cells. Ablation of Nox5α by small interfering RNAs abrogated ROS production, inhibited cellular activities, including proliferation, migration, and survival, and suppressed tumorigenicity in immunodeficient NOG mice. The findings suggest that Nox5α is a key molecule for redox-signal-mediated maintenance of the HTLV-1 transformation phenotype and could be a potential molecular target for therapeutic intervention in cancer development. IMPORTANCE: HTLV-1 is the first human oncogenic retrovirus shown to be associated with ATL. Despite the extensive study over the years, the mechanism underlying HTLV-1-induced cell transformation is not fully understood. In this study, we addressed the expression and function of ROS-generating Nox family genes in HTLV-1-transformed cells. Our report provides the first evidence that the upregulated expression of Nox5α is associated with the pathological state of ATL peripheral blood mononuclear cells and that Nox5α is an integral component of the Jak-STAT5 signaling pathway in HTLV-1-transformed T cells. Nox5α-derived ROS are critically involved in the regulation of cellular activities, including proliferation, migration, survival, and tumorigenicity, in HTLV-1-transformed cells. These results indicate that Nox5α-derived ROS are functionally required for maintenance of the HTLV-1 transformation phenotype. The finding provides new insight into the redox-dependent mechanism of HTLV-1 transformation and raises an intriguing possibility that Nox5α serves as a potential molecular target to treat HTLV-1-related leukemia.


Asunto(s)
Transformación Celular Viral/genética , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Leucemia-Linfoma de Células T del Adulto/patología , Proteínas de la Membrana/metabolismo , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Acetilcisteína/farmacología , Línea Celular Transformada , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/genética , Transformación Celular Neoplásica/genética , Humanos , Interleucina-2/metabolismo , Quinasas Janus/metabolismo , Leucemia-Linfoma de Células T del Adulto/virología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , NADPH Oxidasa 5 , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/genética , Compuestos Onio/farmacología , Interferencia de ARN , ARN Interferente Pequeño , Factor de Transcripción STAT5/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Regulación hacia Arriba
5.
Arterioscler Thromb Vasc Biol ; 35(11): 2316-25, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26404487

RESUMEN

OBJECTIVE: Although immune responses drive the pathogenesis of atherosclerosis, mechanisms that control antigen-presenting cell (APC)-mediated immune activation in atherosclerosis remain elusive. We here investigated the function of hypoxia-inducible factor (HIF)-1α in APCs in atherosclerosis. APPROACH AND RESULTS: We found upregulated HIF1α expression in CD11c(+) APCs within atherosclerotic plaques of low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice. Conditional deletion of Hif1a in CD11c(+) APCs in high-fat diet-fed Ldlr(-/-) mice accelerated atherosclerotic plaque formation and increased lesional T-cell infiltrates, revealing a protective role of this transcription factor. HIF1α directly controls Signal Transducers and Activators of Transcription 3 (Stat3), and a reduced STAT3 expression was found in HIF1α-deficient APCs and aortic tissue, together with an upregulated interleukin-12 expression and expansion of type 1 T-helper (Th1) cells. Overexpression of STAT3 in Hif1a-deficient APCs in bone marrow reversed enhanced atherosclerotic lesion formation and reduced Th1 cell expansion in chimeric Ldlr(-/-) mice. Notably, deletion of Hif1a in LysM(+) bone marrow cells in Ldlr(-/-) mice did not affect lesion formation or T-cell activation. In human atherosclerotic lesions, HIF1α, STAT3, and interleukin-12 protein were found to colocalize with APCs. CONCLUSIONS: Our findings identify HIF1α to antagonize APC activation and Th1 T cell polarization during atherogenesis in Ldlr(-/-) mice and to attenuate the progression of atherosclerosis. These data substantiate the critical role of APCs in controlling immune mechanisms that drive atherosclerotic lesion development.


Asunto(s)
Células Presentadoras de Antígenos/metabolismo , Aorta/metabolismo , Enfermedades de la Aorta/metabolismo , Aterosclerosis/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/deficiencia , Linfocitos T Colaboradores-Inductores/metabolismo , Animales , Células Presentadoras de Antígenos/inmunología , Aorta/inmunología , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/patología , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/patología , Antígeno CD11c/genética , Antígeno CD11c/metabolismo , Enfermedades de las Arterias Carótidas/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Interleucina-12/metabolismo , Activación de Linfocitos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Placa Aterosclerótica , Receptores de LDL/deficiencia , Receptores de LDL/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Linfocitos T Colaboradores-Inductores/inmunología
6.
J Enzyme Inhib Med Chem ; 30(5): 689-721, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25347767

RESUMEN

The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation-deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009-2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [(18)F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (<1% O(2)), and the unfolded protein response (UPR) activated by endoplasmatic reticulum (ER) stress and operating at more severe hypoxia (<0.2%). The prioritised targets were the HIF-regulated proteins carbonic anhydrase IX (CAIX), the lactate transporter MCT4 and the PERK/eIF2α/ATF4-arm of the UPR. The METOXIA project has developed patented compounds targeting CAIX with a preclinical documented effect. Since hypoxia-specific treatments alone are not curative they will have to be combined with traditional anti-cancer therapy to eradicate the aerobic cancer cell population as well.


Asunto(s)
Descubrimiento de Drogas , Neoplasias/tratamiento farmacológico , Animales , Hipoxia de la Célula/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Metástasis de la Neoplasia/tratamiento farmacológico , Metástasis de la Neoplasia/patología , Neoplasias/patología , Relación Estructura-Actividad
7.
J Cell Sci ; 125(Pt 4): 956-64, 2012 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-22399808

RESUMEN

Urotensin-II (U-II) has been considered as one of the most potent vasoactive peptides, although its physiological and pathophysiological role is still not finally resolved. Recent evidence suggests that it promotes angiogenic responses in endothelial cells, although the underlying signalling mechanisms are unclear. Reactive oxygen species derived from NADPH oxidases are major signalling molecules in the vasculature. Because NOX2 is functional in endothelial cells, we investigated the role of the NOX2-containing NADPH oxidase in U-II-induced angiogenesis and elucidated a possible contribution of hypoxia-inducible factor-1 (HIF-1), the master regulator of hypoxic angiogenesis, in the response to U-II. We found that U-II increases angiogenesis in vitro and in vivo, and these responses were prevented by antioxidants, NOX2 knockdown and in Nox2(-/-) mice. In addition, U-II-induced angiogenesis was dependent on HIF-1. Interestingly, U-II increased NOX2 transcription involving HIF-1, and chromatin immunoprecipitation confirmed NOX2 as a target gene of HIF-1. In support, NOX2 levels were greatly diminished in U-II-stimulated isolated vessels derived from mice deficient in endothelial HIF-1. Conversely, reactive oxygen species derived from NOX2 were required for U-II activation of HIF and upregulation of HIF-1. In line with this, U-II-induced upregulation of HIF-1 was absent in Nox2(-/-) vessels. Collectively, these findings identified HIF-1 and NOX2 as partners acting in concert to promote angiogenesis in response to U-II. Because U-II has been found to be elevated in cardiovascular disorders and in tumour tissues, this feed-forward mechanism could be an interesting anti-angiogenic therapeutic option in these disorders.


Asunto(s)
Factor 1 Inducible por Hipoxia/metabolismo , Glicoproteínas de Membrana/metabolismo , NADPH Oxidasas/metabolismo , Neovascularización Fisiológica , Urotensinas/metabolismo , Animales , Retroalimentación Fisiológica , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , NADPH Oxidasa 2 , NADPH Oxidasas/biosíntesis , NADPH Oxidasas/deficiencia , NADPH Oxidasas/genética , Neovascularización Patológica , Neovascularización Fisiológica/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Urotensinas/farmacología
8.
Blood ; 119(5): 1292-301, 2012 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-22144179

RESUMEN

The hypoxia-inducible transcription factor-1α (HIF-1α) is a major regulator of angiogenesis, carcinogenesis, and various processes by which cells adapt to hypoxic conditions. Therefore, the identification of critical players regulating HIF-1α is not only important for the understanding of angiogenesis and different cancer phenotypes, but also for unraveling new therapeutic options. We report a novel mechanism by which HIF-1α is degraded after glycogen synthase kinase-3 (GSK-3)-induced phosphorylation and recruitment of the ubiquitin ligase and tumor suppressor F-box and WD protein Fbw7. Further, experiments with GSK-3ß and Fbw7-deficient cells revealed that GSK-3ß and Fbw7-dependent HIF-1α degradation can be antagonized by ubiquitin-specific protease 28 (USP28). In agreement with this, Fbw7 and USP28 reciprocally regulated cell migration and angiogenesis in an HIF-1α-dependent manner. Therefore, we have identified a new pathway that could be targeted at the level of GSK-3, Fbw7, or USP28 to influence HIF-1α-dependent processes like angiogenesis and metastasis.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Movimiento Celular/genética , Proliferación Celular , Proteínas F-Box/metabolismo , Glucógeno Sintasa Quinasa 3/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neovascularización Fisiológica/genética , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Ciclo Celular/fisiología , Proteínas F-Box/fisiología , Proteína 7 que Contiene Repeticiones F-Box-WD , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Células HCT116 , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Procesamiento Proteico-Postraduccional/genética , Proteolisis , Transducción de Señal/genética , Ubiquitina Tiolesterasa/fisiología , Ubiquitina-Proteína Ligasas/fisiología
9.
J Biol Chem ; 287(7): 4835-52, 2012 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-22157766

RESUMEN

Here, we report that activation of different types of tissue macrophages, including microglia, by lipopolysaccharide (LPS) or GM-CSF stimulation correlates with the quantitative redistribution of NADPH oxidase (cyt b(558)) from the plasma membrane to an intracellular stimulus-responsive storage compartment. Cryo-immunogold labeling of gp91(phox) and CeCl(3) cytochemistry showed the presence of gp91(phox) and oxidant production in numerous small (<100 nm) vesicles. Cell homogenization and sucrose gradient centrifugation in combination with transferrin-HRP/DAB ablation showed that more than half of cyt b(558) is present in fractions devoid of endosomal markers, which is supported by morphological evidence to show that the cyt b(558)-containing compartment is distinct from endosomes or biosynthetic organelles. Streptolysin-O-mediated guanosine 5'-3-O-(thio)triphosphate loading of Ra2 microglia caused exocytosis of a major complement of cyt b(558) under conditions where lysosomes or endosomes were not mobilized. We establish phagocytic particles and soluble mediators ATP, TNFα, and CD40L as physiological inducers of cyt b(558) exocytosis to the cell surface, and by shRNA knockdown, we identify Rab27A/B as positive or negative regulators of vesicular mobilization to the phagosome or the cell surface, respectively. Exocytosis was followed by clathrin-dependent internalization of cyt b(558), which could be blocked by a dominant negative mutant of the clathrin-coated pit-associated protein Eps15. Re-internalized cyt b(558) did not reach lysosomes but associated with recycling endosomes and undefined vesicular elements. In conclusion, cyt b(558) depends on clathrin for internalization, and in activated macrophages NADPH oxidase occupies a Rab27A/B-regulated secretory compartment, which allows rapid agonist-induced redistribution of superoxide production in the cell.


Asunto(s)
Vesículas Cubiertas por Clatrina/enzimología , Activación de Macrófagos/fisiología , Macrófagos/enzimología , Glicoproteínas de Membrana/metabolismo , NADPH Oxidasas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Animales , Ligando de CD40/genética , Ligando de CD40/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Células Cultivadas , Clatrina/genética , Clatrina/metabolismo , Vesículas Cubiertas por Clatrina/genética , Grupo Citocromo b/genética , Grupo Citocromo b/metabolismo , Endosomas/enzimología , Endosomas/genética , Exocitosis/fisiología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos/citología , Glicoproteínas de Membrana/genética , Microglía/enzimología , NADPH Oxidasa 2 , NADPH Oxidasas/genética , Ratas , Superóxidos/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas rab27 de Unión a GTP
10.
iScience ; 25(1): 103596, 2022 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-34988410

RESUMEN

Childhood-onset myocardial hypertrophy and cardiomyopathic changes are associated with significant morbidity and mortality in early life, particularly in patients with Noonan syndrome, a multisystemic genetic disorder caused by autosomal dominant mutations in genes of the Ras-MAPK pathway. Although the cardiomyopathy associated with Noonan syndrome (NS-CM) shares certain cardiac features with the hypertrophic cardiomyopathy caused by mutations in sarcomeric proteins (HCM), such as pathological myocardial remodeling, ventricular dysfunction, and increased risk for malignant arrhythmias, the clinical course of NS-CM significantly differs from HCM. This suggests a distinct pathophysiology that remains to be elucidated. Here, through analysis of sarcomeric myosin conformational states, histopathology, and gene expression in left ventricular myocardial tissue from NS-CM, HCM, and normal hearts complemented with disease modeling in cardiomyocytes differentiated from patient-derived PTPN11 N308S/+ induced pluripotent stem cells, we demonstrate distinct disease phenotypes between NS-CM and HCM and uncover cell cycle defects as a potential driver of NS-CM.

11.
Nat Cardiovasc Res ; 1(2): 157-173, 2022 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-39195995

RESUMEN

Clinical presentation of congenital heart disease is heterogeneous, making identification of the disease-causing genes and their genetic pathways and mechanisms of action challenging. By using in vivo electrocardiography, transthoracic echocardiography and microcomputed tomography imaging to screen 3,894 single-gene-null mouse lines for structural and functional cardiac abnormalities, here we identify 705 lines with cardiac arrhythmia, myocardial hypertrophy and/or ventricular dilation. Among these 705 genes, 486 have not been previously associated with cardiac dysfunction in humans, and some of them represent variants of unknown relevance (VUR). Mice with mutations in Casz1, Dnajc18, Pde4dip, Rnf38 or Tmem161b genes show developmental cardiac structural abnormalities, with their human orthologs being categorized as VUR. Using UK Biobank data, we validate the importance of the DNAJC18 gene for cardiac homeostasis by showing that its loss of function is associated with altered left ventricular systolic function. Our results identify hundreds of previously unappreciated genes with potential function in congenital heart disease and suggest causal function of five VUR in congenital heart disease.

12.
Physiol Genomics ; 43(2): 87-98, 2011 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-20978110

RESUMEN

Disordered angiogenesis is implicated in pulmonary vascular remodeling secondary to congenital heart diseases (CHD). However, the underlying genes are not well delineated. We showed previously that an ovine model of CHD with increased pulmonary blood flow (PBF, Shunt) has an "angiogenesis burst" between 1 and 4 wk of age. Thus we hypothesized that the increased PBF elicited a proangiogenic gene expression profile before onset of vessel growth. To test this we utilized microarray analysis to identify genes that could be responsible for the angiogenic response. Total RNA was isolated from lungs of Shunt and control lambs at 3 days of age and hybridized to Affymetrix gene chips for microarray analyses (n = 8/group). Eighty-nine angiogenesis-related genes were found to be upregulated and 26 angiogenesis-related genes downregulated in Shunt compared with control lungs (cutting at 1.2-fold difference, P < 0.05). We then confirmed upregulation of proangiogenic genes FGF2, Angiopoietin2 (Angpt2), and Birc5 at mRNA and protein levels and upregulation of ccl2 at mRNA level in 3-day Shunt lungs. Furthermore, we found that pulmonary arterial endothelial cells (PAEC) isolated from fetal lambs exhibited increased expression of FGF2, Angpt2, Birc5, and ccl2 and enhanced angiogenesis when exposed to elevated shear stress (35 dyn/cm²) compared with cells exposed to more physiological shear stress (20 dyn/cm²). Finally, we demonstrated that blocking FGF2, Angpt2, Birc5, or ccl2 signaling with neutralizing antibodies or small interfering RNA (siRNA) significantly decreased the angiogenic response induced by shear stress. In conclusion, we have identified a "proangiogenic" gene expression profile in a lamb model of CHD with increased PBF that precedes onset of pulmonary vascular remodeling. Our data indicate that FGF2, Angpt2, Birc5, and ccl2 may play important roles in the angiogenic response.


Asunto(s)
Perfilación de la Expresión Génica , Cardiopatías/congénito , Cardiopatías/genética , Pulmón/irrigación sanguínea , Pulmón/fisiopatología , Neovascularización Fisiológica/genética , Circulación Pulmonar/genética , Angiopoyetina 2/genética , Angiopoyetina 2/metabolismo , Animales , Western Blotting , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo/genética , Células Endoteliales/metabolismo , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Cardiopatías/patología , Cardiopatías/fisiopatología , Hemodinámica/fisiología , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Fenotipo , Arteria Pulmonar/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ovinos , Estrés Mecánico , Regulación hacia Arriba/genética
13.
J Biol Chem ; 285(34): 26182-9, 2010 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-20566631

RESUMEN

During tumor progression, malignant cells must repeatedly survive microenvironmental stress. Hypoxia-inducible factor-1 (HIF-1) signaling has emerged as one major pathway allowing cellular adaptation to stress. Recent findings led to the hypothesis that HIF-1alpha may enhance the metastatic potential of tumor cells by a survival-independent mechanism. So far it has not been shown that HIF-1alpha also directly regulates invasive processes during metastasis in addition to conferring a survival advantage to metastasizing tumor cells. In a hypoxia-tolerant tumor cell line (L-CI.5s), which did not rely on HIF-1 signaling for viability in vitro and in vivo, knockdown of Hif-1alpha reduced invasiveness of the tumor cells in vitro as well as extravasation and secondary infiltration in vivo. Liver metastases associated induction of proinvasive receptor tyrosine kinase Met phosphorylation as well as gelatinolytic activity were Hif-1alpha-dependent. Indeed, promoter activity of the matrix metalloproteinase-9 (mmp-9) was shown to be Hif-1alpha-dependent. This study uncovers a new survival-independent biological function of HIF-1alpha contributing to the efficacy of metastases formation.


Asunto(s)
Hipoxia de la Célula , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Metástasis de la Neoplasia/patología , Animales , Línea Celular Tumoral , Supervivencia Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/secundario , Metaloproteinasa 9 de la Matriz/genética , Ratones , Proteínas Tirosina Quinasas Receptoras/metabolismo
14.
Circ Res ; 104(10): 1169-77, 2009 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-19390057

RESUMEN

Cyclic nucleotide phosphodiesterases (PDEs) control the levels of the second messengers cAMP and cGMP in many cell types including endothelial cells. Although PDE2 has the unique property to be activated by cGMP but to hydrolyze cAMP, its role in endothelial function is only poorly understood. Reactive oxygen species (ROS) have been recognized as signaling molecules controlling many endothelial functions. We thus investigated whether PDE2 would link to ROS generation and proliferative responses in human umbilical vein endothelial cells in response to thrombin. Thrombin stimulated the GTPase Rac1, known to activate NADPH oxidases, and enhanced ROS formation, whereas PDE2 inhibition or depletion by short hairpin (sh)RNA prevented these responses. Similar observations were made with 8-Br-cGMP or atrial natriuretic peptide. In agreement, thrombin elevated cGMP but decreased cAMP levels, whereas db-cAMP or forskolin diminished Rac1 activity and ROS production. Subsequently, PDE2 overexpression activated Rac1, increased ROS generation, and enhanced proliferation and in vitro capillary formation. These responses were not observed in the presence of inactive Rac1 or shRNA against the NADPH oxidase subunit NOX2. Inhibition or depletion of PDE2 also prevented thrombin-induced proliferation and capillary formation. Importantly, downregulation of PDE2 by lentiviral shRNA or PDE2 inhibition prevented vessel sprouting from mouse aortic explants and in vivo angiogenesis in a mouse model, respectively. In summary, PDE2 promotes activation of NADPH oxidase-dependent ROS production and subsequent endothelial proliferation and angiogenesis. Targeting PDE2 may provide a new therapeutic approach in diseases associated with endothelial dysfunction, oxidative stress, vascular proliferation, and angiogenesis.


Asunto(s)
Proliferación Celular , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/metabolismo , Endotelio Vascular/metabolismo , Glicoproteínas de Membrana/metabolismo , NADPH Oxidasas/metabolismo , Neovascularización Fisiológica/fisiología , Trombina/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Aorta/citología , Aorta/metabolismo , Células Cultivadas , GMP Cíclico/metabolismo , Endotelio Vascular/citología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , NADPH Oxidasa 2 , Especies Reactivas de Oxígeno/metabolismo , Venas Umbilicales/citología , Venas Umbilicales/metabolismo , Quinasas p21 Activadas/metabolismo
15.
J Clin Med ; 10(22)2021 Nov 11.
Artículo en Inglés | MEDLINE | ID: mdl-34830538

RESUMEN

Hypertrophic cardiomyopathy (HCM) is associated with adverse left ventricular (LV) remodeling causing dysfunction and malignant arrhythmias. Severely affected patients present with disease onset during childhood and sudden cardiac death risk (SCD) stratification is of the highest importance in this cohort. This study aimed to investigate genotype-phenotype association regarding clinical outcome and disease progression in pediatric onset HCM. Medical charts from forty-nine patients with pediatric HCM who had undergone genetic testing were reviewed for retrospective analysis. Demographic, clinical, transthoracic echocardiographic, electrocardiographic, long-term electrocardiogram, cardiopulmonary exercise test, cardiac magnetic resonance, and medication data were recorded. Childhood onset HCM was diagnosed in 29 males and 20 females. Median age at last follow-up was 18.7 years (range 2.6-51.7 years) with a median follow-up time since diagnosis of 8.5 years (range 0.2-38.0 years). Comparison of patients carrying mutations in distinct genes and comparison of genotype-negative with genotype-positive individuals, revealed no differences in functional classification, LV morphology, hypertrophy, systolic and diastolic function, fibrosis and cardiac medication. Patients with compound mutations had a significantly higher risk for major arrhythmic events than a single-mutation carrier. No association between affected genes and disease severity or progression was identified in this cohort.

16.
Cardiovasc Diagn Ther ; 11(6): 1295-1309, 2021 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-35070799

RESUMEN

BACKGROUND: Right ventricular impairment (RVI) secondary to altered hemodynamics contributes to morbidity and mortality in adult patients after tetralogy of Fallot (TOF) repair. The goal of this study was to describe signaling pathways contributing to right ventricular (RV) remodeling by analyzing over lifetime alterations of RV gene expression in affected patients. METHODS: RV tissue was collected at the time of cardiac surgery in 13 patients with a diagnosis of TOF. RNA was isolated and whole transcriptome sequencing was performed. Gene profiles were compared between a group of 6 adults with signs of RVI undergoing right ventricle to pulmonary artery conduit surgery and a group of 7 infants, undergoing TOF correction. Definition of RVI in adult patients was based on clinical symptoms, evidence of RV hypertrophy, dilation, dysfunction or elevated pressure on echocardiographic, cardiovascular magnetic resonance, or catheterization evaluation. RESULTS: Median age was 34 years in RVI patients and 5 months in infants. Based on P adjusted value <0.01, RNA sequencing of RV specimens identified a total of 3,010 differentially expressed genes in adult patients with TOF and RVI as compared to infant patients with TOF. Gene Ontology and Kyoto Encyclopedia of Genes databases highlighted pathways involved in cellular metabolism, cell-cell communication, cell cycling and cellular contractility to be dysregulated in adults with corrected TOF and chronic RVI. CONCLUSIONS: RV transcriptome profiling in adult patients with RVI after TOF repair allows identification of signaling pathways, contributing to pathologic RV remodeling and helps in the discovery of biomarkers for disease progression and of new therapeutic targets.

17.
Mol Cell Biol ; 27(9): 3253-65, 2007 May.
Artículo en Inglés | MEDLINE | ID: mdl-17325032

RESUMEN

Hypoxia-inducible transcription factor 1alpha (HIF-1alpha) is a key player in the response to hypoxia. Additionally, HIF-1alpha responds to growth factors and hormones which can act via protein kinase B (Akt). However, HIF-1alpha is not a direct substrate for this kinase. Therefore, we investigated whether the protein kinase B target glycogen synthase kinase 3 (GSK-3) may have an impact on HIF-1alpha. We found that the inhibition or depletion of GSK-3 induced HIF-1alpha whereas the overexpression of GSK-3beta reduced HIF-1alpha. These effects were mediated via three amino acid residues in the oxygen-dependent degradation domain of HIF-1alpha. In addition, mutation analyses and experiments with von Hippel-Lindau (VHL)-defective cells indicated that GSK-3 mediates HIF-1alpha degradation in a VHL-independent manner. In line with these observations, the inhibition of the proteasome reversed the GSK-3 effects, indicating that GSK-3 may target HIF-1alpha to the proteasome by phosphorylation. Thus, the direct regulation of HIF-1alpha stability by GSK-3 may influence physiological processes or pathophysiological situations such as metabolic diseases or tumors.


Asunto(s)
Glucógeno Sintasa Quinasa 3/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Humanos , Hidroxilación , Insulina/farmacología , Cloruro de Litio/farmacología , Mutación/genética , Fosforilación , Inhibidor 1 de Activador Plasminogénico/genética , Regiones Promotoras Genéticas/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , ARN Interferente Pequeño/genética , Elementos de Respuesta , Activación Transcripcional/genética , Ubiquitina/metabolismo
18.
Mol Biol Cell ; 18(12): 4691-7, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17898080

RESUMEN

The oxygen sensitive alpha-subunit of the hypoxia-inducible factor-1 (HIF-1) is a major trigger of the cellular response to hypoxia. Although the posttranslational regulation of HIF-1alpha by hypoxia is well known, its transcriptional regulation by hypoxia is still under debate. We, therefore, investigated the regulation of HIF-1alpha mRNA in response to hypoxia in pulmonary artery smooth muscle cells. Hypoxia rapidly enhanced HIF-1alpha mRNA levels and HIF-1alpha promoter activity. Furthermore, inhibition of the phosphatidylinositol 3-kinase (PI3K)/AKT but not extracellular signal-regulated kinase 1/2 pathway blocked the hypoxia-dependent induction of HIF-1alpha mRNA and HIF-1alpha promoter activity, suggesting involvement of a PI3K/AKT-regulated transcription factor. Interestingly, hypoxia also induced nuclear factor-kappaB (NFkappaB) nuclear translocation and activity. In line, expression of the NFkappaB subunits p50 and p65 enhanced HIF-1alpha mRNA levels, whereas blocking of NFkappaB by an inhibitor of nuclear factor-kappaB attenuated HIF-1alpha mRNA induction by hypoxia. Reporter gene assays revealed the presence of an NFkappaB site within the HIF-1alpha promoter, and mutation of this site abolished induction by hypoxia. In line, gel shift analysis and chromatin immunoprecipitation confirmed binding of p50 and p65 NFkappaB subunits to the HIF-1alpha promoter under hypoxia. Together, these findings provide a novel mechanism in which hypoxia induces HIF-1alpha mRNA expression via the PI3K/AKT pathway and activation of NFkappaB.


Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/metabolismo , Miocitos del Músculo Liso/metabolismo , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transcripción Genética/genética , Regulación hacia Arriba/genética , Animales , Arterias/metabolismo , Secuencia de Bases , Células Cultivadas , Humanos , Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-akt , ARN Mensajero/genética , Transducción de Señal
19.
Redox Biol ; 34: 101536, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32413743

RESUMEN

Cardiovascular side effects are frequent problems accompanying systemic glucocorticoid therapy, although the underlying mechanisms are not fully resolved. Reactive oxygen species (ROS) have been shown to promote various cardiovascular diseases although the link between glucocorticoid and ROS signaling has been controversial. As the family of NADPH oxidases has been identified as important source of ROS in the cardiovascular system we investigated the role of NADPH oxidases in response to the synthetic glucocorticoid dexamethasone in the cardiovascular system in vitro and in vivo in mice lacking functional NADPH oxidases due to a mutation in the gene coding for the essential NADPH oxidase subunit p22phox. We show that dexamethasone induced NADPH oxidase-dependent ROS generation, leading to vascular proliferation and angiogenesis due to activation of the transcription factor hypoxia-inducible factor-1 (HIF1). Chronic treatment of mice with low doses of dexamethasone resulted in the development of systemic hypertension, cardiac hypertrophy and left ventricular dysfunction, as well as in pulmonary hypertension and pulmonary vascular remodeling. In contrast, mice deficient in p22phox-dependent NADPH oxidases were protected against these cardiovascular side effects. Mechanistically, dexamethasone failed to upregulate HIF1α levels in these mice, while vascular HIF1α deficiency prevented pulmonary vascular remodeling. Thus, p22phox-dependent NADPH oxidases and activation of the HIF pathway are critical elements in dexamethasone-induced cardiovascular pathologies and might provide interesting targets to limit cardiovascular side effects in patients on chronic glucocorticoid therapy.


Asunto(s)
Cardiopatías , Hipertensión Pulmonar , Animales , Glucocorticoides , Humanos , Hipertensión Pulmonar/inducido químicamente , Factor 1 Inducible por Hipoxia , Ratones , NADPH Oxidasas/genética , Especies Reactivas de Oxígeno
20.
Am J Physiol Endocrinol Metab ; 297(3): E735-48, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19549791

RESUMEN

Obesity is associated with a state of chronic low-grade inflammation. Immune cells accumulate in white adipose tissue (WAT). The vascular endothelium plays an interactive role in these infiltration and inflammatory processes. Mature and hypertrophic adipocytes are considered as the major adipogenic cell type secreting proinflammatory cytokines in WAT. In contrast, the proinflammatory capacity of preadipocytes and their role in endothelial cell activation have been neglected so far. To gain new insights into this molecular and cellular cross-talk, we examined the proinflammatory expression and secretion of normoxia, hypoxia, and TNFalpha-treated human preadipocytes and adipocytes (SGBS cells) and their impact on human microvascular endothelial cell (HMEC-1) function. In this study, stimulation of HMEC-1 with conditioned media (CM) from preadipocytes increased endothelial ICAM-1 expression and monocyte adhesion but not adipocyte-CM. After hypoxia and TNFalpha stimulation of SGBS cells, adipocyte-CM induced and preadipocyte-CM enhanced the monocyte adhesion. Concordantly, the expression of proinflammatory adipokines was considerably higher in preadipocytes than in adipocytes. SGBS-CM upregulated the phosphorylation of three MAPK pathways, STAT1/3, and c-Jun in HMEC-1, whereas the NF-kappaB pathway was not affected. Inhibitor experiments showed that monocyte/endothelial cell-cell adhesion and endothelial ICAM-1 expression was JNK and JAK-1/STAT1/3 pathway dependent and revealed IL-6 as a major mediator in CM increasing monocyte/endothelial cell-cell adhesion via the STAT1/3 pathway. Our study shows that preadipocytes rather than adipocytes operate as potent activators of endothelial cells. This can be enhanced in preadipocytes and induced in adipocytes by TNFalpha and hypoxia in a manner similar to what may occur in WAT in the etiology of obesity.


Asunto(s)
Adipocitos/efectos de los fármacos , Adipocitos/fisiología , Células Endoteliales/fisiología , Oxígeno/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Adipocitos/metabolismo , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Hipoxia de la Célula/genética , Hipoxia de la Célula/fisiología , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Perfilación de la Expresión Génica , Humanos , Proteínas I-kappa B/metabolismo , Mediadores de Inflamación/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Monocitos/efectos de los fármacos , Monocitos/fisiología , Inhibidor NF-kappaB alfa , Fosforilación/efectos de los fármacos , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Células Madre/fisiología , Factor de Transcripción ReIA/metabolismo , Células U937
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