RESUMO
RATIONALE: Innate and adaptive immune responses alter numerous homeostatic processes that are controlled by nuclear hormone receptors. NR4A1 is a nuclear receptor that is induced in vascular pathologies, where it mediates protection. OBJECTIVE: The underlying mechanisms that regulate the activity of NR4A1 during vascular injury are not clear. We therefore searched for modulators of NR4A1 function that are present during vascular inflammation. METHODS AND RESULTS: We report that the protein encoded by interferon stimulated gene 12 (ISG12), is a novel interaction partner of NR4A1 that inhibits the transcriptional activities of NR4A1 by mediating its Crm1-dependent nuclear export. Using 2 models of vascular injury, we show that ISG12-deficient mice are protected from neointima formation. This effect is dependent on the presence of NR4A1, as mice deficient for both ISG12 and NR4A1 exhibit neointima formation similar to wild-type mice. CONCLUSIONS: These findings identify a previously unrecognized feedback loop activated by interferons that inhibits the vasculoprotective functions of NR4A nuclear receptors, providing a potential new therapeutic target for interferon-driven pathologies.
Assuntos
Lesões das Artérias Carótidas/prevenção & controle , Artéria Femoral/metabolismo , Inflamação/prevenção & controle , Proteínas de Membrana/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Proteínas/metabolismo , Lesões do Sistema Vascular/prevenção & controle , Transporte Ativo do Núcleo Celular , Animais , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/imunologia , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Retroalimentação Fisiológica , Artéria Femoral/lesões , Artéria Femoral/patologia , Regulação da Expressão Gênica , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Interferons/metabolismo , Carioferinas/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/lesões , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas/genética , Interferência de RNA , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Tempo , Transcrição Gênica , Transfecção , Lesões do Sistema Vascular/genética , Lesões do Sistema Vascular/imunologia , Lesões do Sistema Vascular/metabolismo , Lesões do Sistema Vascular/patologia , Proteína Exportina 1RESUMO
During embryonic development, lymph sacs form from the cardinal vein, and sprout centrifugally to form mature lymphatic networks. Separation of the lymphatic from the blood circulation by a hitherto unknown mechanism is essential for the homeostatic function of the lymphatic system. O-glycans on the lymphatic endothelium have recently been suggested to be required for establishment and maintenance of distinct blood and lymphatic systems, primarily by mediating proper function of podoplanin. Here, we show that this separation process critically involves platelet activation by podoplanin. We found that platelet aggregates build up in wild-type embryos at the separation zone of podoplanin(+) lymph sacs and cardinal veins, but not in podoplanin(-/-) embryos. Thus, podoplanin(-/-) mice develop a "nonseparation" phenotype, characterized by a blood-filled lymphatic network after approximately embryonic day 13.5, which, however, partially resolves in postnatal mice. The same embryonic phenotype is also induced by treatment of pregnant mice with acetyl salicylic acid, podoplanin-blocking antibodies, or by inactivation of the kindlin-3 gene required for platelet aggregation. Therefore, interaction of endothelial podoplanin of the developing lymph sac with circulating platelets from the cardinal vein is critical for separating the lymphatic from the blood vascular system.
Assuntos
Plaquetas/fisiologia , Vasos Sanguíneos/embriologia , Vasos Linfáticos/embriologia , Glicoproteínas de Membrana/fisiologia , Animais , Anti-Infecciosos/farmacologia , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/metabolismo , Proteínas do Citoesqueleto/fisiologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Endotélio Linfático/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas Imunoenzimáticas , Vasos Linfáticos/efeitos dos fármacos , Vasos Linfáticos/metabolismo , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Agregação Plaquetária , Gravidez , Ácido Salicílico/farmacologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/fisiologiaRESUMO
OBJECTIVE: Our goal was to examine the influence of indirubin-3'-monoxime (I3MO), a natural product-derived cyclin-dependent kinase inhibitor, on vascular smooth muscle cell (VSMC) proliferation in vitro, experimentally induced neointima formation in vivo, and related cell signaling pathways. METHODS AND RESULTS: I3MO dose-dependently inhibited platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation by arresting cells in the G(0)/G(1) phase of the cell cycle as assessed by 5-bromo-2'-deoxyuridine incorporation and flow cytometry. PDGF-induced activation of the kinases Akt, Erk1/2, and p38(MAPK) was not affected. In contrast, I3MO specifically blocked PDGF-, interferon-γ-, and thrombin-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3). Human endothelial cells (EA.hy926) responded to I3MO with increased endothelial nitric oxide synthase activity as assessed via [(14)C]l-arginine/[(14)C]l-citrulline conversion. The specific STAT3 inhibitor Stattic led to decreased VSMC proliferation, and transient expression of a constitutively active form of STAT3 overcame the I3MO-induced cell cycle arrest in mouse embryonic fibroblasts. In a murine femoral artery cuff model, I3MO prevented neointima formation while reducing STAT3 phosphorylation and the amount of proliferating Ki67-positive cells. CONCLUSIONS: I3MO represses PDGF- and thrombin-induced VSMC proliferation and, in vivo, neointima formation, likely because it specifically blocks STAT3 signaling. This profile and its positive effect on endothelial NO production turns I3MO into a promising lead compound to prevent restenosis.
Assuntos
Arteriopatias Oclusivas/prevenção & controle , Proliferação de Células , Indóis/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Oximas/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Arteriopatias Oclusivas/metabolismo , Arteriopatias Oclusivas/patologia , Becaplermina , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Constrição Patológica , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Artéria Femoral/efeitos dos fármacos , Artéria Femoral/metabolismo , Artéria Femoral/patologia , Citometria de Fluxo , Humanos , Hiperplasia , Interferon gama/metabolismo , Antígeno Ki-67/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Fosforilação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-sis , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Trombina/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
The developing sinus venosus myocardium, derived from the posterior heart field, contributes to the atrial septum, the posterior atrial wall, the sino-atrial node, and myocardium lining the pulmonary and cardinal veins, all expressing podoplanin, a coelomic and myocardial marker. We compared development and differentiation of the myocardium and vascular wall of the pulmonary veins (PV), left atrial dorsal wall, and atrial septum in wild type with podoplanin knockout mouse embryos (E10.5-E18.5) by 3D reconstruction and immunohistochemistry. Expression of Nkx2.5 in the pulmonary venous myocardium changes from mosaic to positive during development pointing out a high proliferative rate compared with Nkx2.5 negative myocardium of the sino-atrial node and cardinal veins. In mutants, myocardium of the PVs, dorsal atrial wall and atrial septum was hypoplastic. The atrial septum and right-sided wall of the PV almost lacked interposed mesenchyme. Extension of smooth muscle cells into the left atrial body was diminished. We conclude that myocardium of the PVs, dorsal atrial wall, and atrial septum, as well as the smooth muscle cells, are derived from the posterior heart field regulated by podoplanin.
Assuntos
Comunicação Interatrial/embriologia , Glicoproteínas de Membrana/deficiência , Músculo Liso Vascular/anormalidades , Miocárdio/patologia , Miócitos de Músculo Liso/patologia , Veias Pulmonares/anormalidades , Actinas/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Idade Gestacional , Átrios do Coração/embriologia , Átrios do Coração/metabolismo , Comunicação Interatrial/metabolismo , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/metabolismo , Imageamento Tridimensional , Imuno-Histoquímica , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Miocárdio/metabolismo , Miócitos de Músculo Liso/metabolismo , Cadeias Leves de Miosina/metabolismo , Organogênese , Veias Pulmonares/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Podoplanin, a mucin-like plasma membrane protein, is expressed by lymphatic endothelial cells and responsible for separation of blood and lymphatic circulation through activation of platelets. Here we show that podoplanin is also expressed by thymic fibroblastic reticular cells (tFRC), a novel thymic medulla stroma cell type associated with thymic conduits, and involved in development of natural regulatory T cells (nTreg). Young mice deficient in podoplanin lack nTreg owing to retardation of CD4(+)CD25(+) thymocytes in the cortex and missing differentiation of Foxp3(+) thymocytes in the medulla. This might be due to CCL21 that delocalizes upon deletion of the CCL21-binding podoplanin from medullar tFRC to cortex areas. The animals do not remain devoid of nTreg but generate them delayed within the first month resulting in Th2-biased hypergammaglobulinemia but not in the death-causing autoimmune phenotype of Foxp3-deficient Scurfy mice.
Assuntos
Fibroblastos/imunologia , Glicoproteínas de Membrana/metabolismo , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Timo/imunologia , Animais , Antígenos CD4/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem da Célula/genética , Células Cultivadas , Quimiocina CCL21/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucinas/metabolismo , Receptor Cross-TalkRESUMO
We investigated the role of podoplanin in development of the sinus venosus myocardium comprising the sinoatrial node, dorsal atrial wall, and primary atrial septum as well as the myocardium of the cardinal and pulmonary veins. We analyzed podoplanin wild-type and knockout mouse embryos between embryonic day 9.5-15.5 using immunohistochemical marker podoplanin; sinoatrial-node marker HCN4; myocardial markers MLC-2a, Nkx2.5, as well as Cx43; coelomic marker WT-1; and epithelial-to-mesenchymal transformation markers E-cadherin and RhoA. Three-dimensional reconstructions were made and myocardial morphometry was performed. Podoplanin mutants showed hypoplasia of the sinoatrial node, primary atrial septum, and dorsal atrial wall. Myocardium lining the wall of the cardinal and pulmonary veins was thin and perforated. Impaired myocardial formation is correlated with abnormal epithelial-to-mesenchymal transformation of the coelomic epithelium due to up-regulated E-cadherin and down-regulated RhoA, which are controlled by podoplanin. Our results demonstrate an important role for podoplanin in development of sinus venosus myocardium.
Assuntos
Átrios do Coração , Glicoproteínas de Membrana , Miocárdio , Nó Sinoatrial , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Epitélio/fisiologia , Feminino , Átrios do Coração/anormalidades , Átrios do Coração/anatomia & histologia , Átrios do Coração/embriologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mesoderma/fisiologia , Camundongos , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologia , Gravidez , Nó Sinoatrial/anormalidades , Nó Sinoatrial/embriologia , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Epicardium and epicardium-derived cells have been shown to be necessary for myocardial differentiation. To elucidate the function of podoplanin in epicardial development and myocardial differentiation, we analyzed podoplanin knockout mouse embryos between embryonic day (E) 9.5 and E15.5 using immunohistochemical differentiation markers, morphometry, and three-dimensional reconstructions. Podoplanin null mice have an increased embryonic lethality, possibly of cardiac origin. Our study reveals impairment in the development of the proepicardial organ, epicardial adhesion, and spreading and migration of the epicardium-derived cells. Mutant embryos show a hypoplastic and perforated compact and septal myocardium, hypoplastic atrioventricular cushions resulting in atrioventricular valve abnormalities, as well as coronary artery abnormalities. The epicardial pathology is correlated with reduced epithelial-mesenchymal transformation caused by up-regulation of E-cadherin, normally down-regulated by podoplanin. Our results demonstrate a role for podoplanin in normal cardiac development based on epicardial-myocardial interaction. Abnormal epicardial differentiation and reduced epithelial-mesenchymal transformation result in deficient epicardium-derived cells leading to myocardial pathology and cardiac anomalies.
Assuntos
Cardiopatias Congênitas/embriologia , Coração/embriologia , Glicoproteínas de Membrana/metabolismo , Miocárdio/metabolismo , Pericárdio/embriologia , Animais , Caderinas/metabolismo , Cardiopatias Congênitas/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Miocárdio/citologia , Pericárdio/citologia , Pericárdio/metabolismoRESUMO
OBJECTIVE: To investigate the mechanisms responsible for the testicular abnormalities and infertility of previously generated male protein C inhibitor (PCI)-deficient mice. DESIGN: Determination of the localization of PCI in the reproductive organs of wild-type males. Generation of double knockout mice lacking the protease inhibitor PCI and one plasminogen activator, either urokinase (uPA) or tissue plasminogen activator (tPA), both of which are PCI-target proteases. SETTING: Animal research and histologic analysis. ANIMAL(S): Male mice of desired genotype. INTERVENTION(S): Fertility testing of double knockout mice. MAIN OUTCOME MEASURE(S): Infertility of PCI(-/-)uPA(-/-) and PCI(-/-)tPA(-/-) double knockout mice. RESULT(S): In the testes of wild-type males PCI was detected in spermatocytes of prophase I, as well as in late spermatids and mature spermatozoa, but absent from somatic cells. All PCI(-/-) uPA(-/-) and PCI(-/-) tPA(-/-) male mice were infertile and histologic analysis of testis showed similar alterations as previously described for PCI(-/-) mice. CONCLUSION(S): The abnormal spermatogenesis of PCI (plasminogen activator inhibitor-3)-deficient mice cannot be rescued by single plasminogen activator knockout.