RESUMO
Resident macrophages densely populate the normal arterial wall, yet their origins and the mechanisms that sustain them are poorly understood. Here we use gene-expression profiling to show that arterial macrophages constitute a distinct population among macrophages. Using multiple fate-mapping approaches, we show that arterial macrophages arise embryonically from CX3CR1(+) precursors and postnatally from bone marrow-derived monocytes that colonize the tissue immediately after birth. In adulthood, proliferation (rather than monocyte recruitment) sustains arterial macrophages in the steady state and after severe depletion following sepsis. After infection, arterial macrophages return rapidly to functional homeostasis. Finally, survival of resident arterial macrophages depends on a CX3CR1-CX3CL1 axis within the vascular niche.
Assuntos
Autorrenovação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Monócitos/citologia , Monócitos/metabolismo , Receptores de Quimiocinas/metabolismo , Animais , Receptor 1 de Quimiocina CX3C , Sobrevivência Celular , Quimiocina CX3CL1/metabolismo , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Imunofenotipagem , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Transgênicos , Fenótipo , Ligação Proteica , Nicho de Células-Tronco , TranscriptomaRESUMO
Human type 2 cytotoxic T (Tc2) cells are enriched in severe eosinophilic asthma and can contribute to airway eosinophilia. PGD2 and its receptor PGD2 receptor 2 (DP2) play important roles in Tc2 cell activation, including migration, cytokine production, and survival. In this study, we revealed novel, to our knowledge, functions of the PGD2/DP2 axis in Tc2 cells to induce tissue-remodeling effects and IgE-independent PGD2 autocrine production. PGD2 upregulated the expression of tissue-remodeling genes in Tc2 cells that enhanced the fibroblast proliferation and protein production required for tissue repair and myofibroblast differentiation. PGD2 stimulated Tc2 cells to produce PGD2 using the routine PGD2 synthesis pathway, which also contributed to TCR-dependent PGD2 production in Tc2 cells. Using fevipiprant, a specific DP2 antagonist, we demonstrated that competitive inhibition of DP2 not only completely blocked the cell migration, adhesion, proinflammatory cytokine production, and survival of Tc2 cells triggered by PGD2 but also attenuated the tissue-remodeling effects and autocrine/paracrine PGD2 production in Tc2 induced by PGD2 and other stimulators. These findings further confirmed the anti-inflammatory effect of fevipiprant and provided a better understanding of the role of Tc2 cells in the pathogenesis of asthma.
Assuntos
Ácidos Indolacéticos/farmacologia , Inflamação/tratamento farmacológico , Prostaglandina D2/antagonistas & inibidores , Piridinas/farmacologia , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Linfócitos T Citotóxicos/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Humanos , Inflamação/imunologia , Prostaglandina D2/biossíntese , Receptores Imunológicos/imunologia , Receptores de Prostaglandina/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
RATIONALE: Inflammation is a key contributor to atherosclerosis. MicroRNA-146a (miR-146a) has been identified as a critical brake on proinflammatory nuclear factor κ light chain enhancer of activated B cells signaling in several cell types, including endothelial cells and bone marrow (BM)-derived cells. Importantly, miR-146a expression is elevated in human atherosclerotic plaques, and polymorphisms in the miR-146a precursor have been associated with risk of coronary artery disease. OBJECTIVE: To define the role of endogenous miR-146a during atherogenesis. METHODS AND RESULTS: Paradoxically, Ldlr-/- (low-density lipoprotein receptor null) mice deficient in miR-146a develop less atherosclerosis, despite having highly elevated levels of circulating proinflammatory cytokines. In contrast, cytokine levels are normalized in Ldlr-/-;miR-146a-/- mice receiving wild-type BM transplantation, and these mice have enhanced endothelial cell activation and elevated atherosclerotic plaque burden compared with Ldlr-/- mice receiving wild-type BM, demonstrating the atheroprotective role of miR-146a in the endothelium. We find that deficiency of miR-146a in BM-derived cells precipitates defects in hematopoietic stem cell function, contributing to extramedullary hematopoiesis, splenomegaly, BM failure, and decreased levels of circulating proatherogenic cells in mice fed an atherogenic diet. These hematopoietic phenotypes seem to be driven by unrestrained inflammatory signaling that leads to the expansion and eventual exhaustion of hematopoietic cells, and this occurs in the face of lower levels of circulating low-density lipoprotein cholesterol in mice lacking miR-146a in BM-derived cells. Furthermore, we identify sortilin-1(Sort1), a known regulator of circulating low-density lipoprotein levels in humans, as a novel target of miR-146a. CONCLUSIONS: Our study reveals that miR-146a regulates cholesterol metabolism and tempers chronic inflammatory responses to atherogenic diet by restraining proinflammatory signaling in endothelial cells and BM-derived cells.
Assuntos
Aterosclerose/metabolismo , Aterosclerose/prevenção & controle , MicroRNAs/metabolismo , Animais , Aterosclerose/patologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Bovinos , VLDL-Colesterol/metabolismo , Dieta Aterogênica/efeitos adversos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Receptores de LDL/metabolismoRESUMO
Artesunate remains the mainstay of treatment for cerebral malaria, but it is less effective in later stages of disease when the host inflammatory response and blood-brain barrier integrity dictate clinical outcomes. Nitric oxide (NO) is an important regulator of inflammation and microvascular integrity, and impaired NO bioactivity is associated with fatal outcomes in malaria. Endogenous NO bioactivity in mammals is largely mediated by S-nitrosothiols (SNOs). Based on these observations, we hypothesized that animals deficient in the SNO-metabolizing enzyme, S-nitrosoglutathione reductase (GSNOR), which exhibit enhanced S-nitrosylation, would have improved outcomes in a preclinical model of cerebral malaria. GSNOR knockout (KO) mice infected with Plasmodium berghei ANKA had significantly delayed mortality compared to WT animals (P < 0.0001), despite higher parasite burdens (P < 0.01), and displayed markedly enhanced survival versus the wild type (WT) when treated with the antimalarial drug artesunate (77% versus 38%; P < 0.001). Improved survival was associated with higher levels of protein-bound NO, decreased levels of CD4+ and CD8+ T cells in the brain, improved blood-brain barrier integrity, and improved coma scores, as well as higher levels of gamma interferon. GSNOR KO animals receiving WT bone marrow had significantly reduced survival following P. berghei ANKA infection compared to those receiving KO bone barrow (P < 0.001). Reciprocal transplants established that survival benefits of GSNOR deletion were attributable primarily to the T cell compartment. These data indicate a role for GSNOR in the host response to malaria infection and suggest that strategies to disrupt its activity will improve clinical outcomes by enhancing microvascular integrity and modulating T cell tissue tropism.
Assuntos
Álcool Desidrogenase/deficiência , Malária Cerebral/patologia , Plasmodium berghei/patogenicidade , Animais , Antimaláricos/administração & dosagem , Artemisininas/administração & dosagem , Artesunato , Modelos Animais de Doenças , Feminino , Malária Cerebral/tratamento farmacológico , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , Análise de Sobrevida , Linfócitos T/imunologia , Resultado do TratamentoRESUMO
OBJECTIVE: Vascular smooth muscle cells (VSMCs) are believed to dedifferentiate and proliferate in response to vessel injury. Recently, adventitial progenitor cells were implicated as a source of VSMCs involved in vessel remodeling. c-Myb is a transcription factor known to regulate VSMC proliferation in vivo and differentiation of VSMCs from mouse embryonic stem cell-derived progenitors in vitro. However, the role of c-Myb in regulating specific adult vascular progenitor cell populations was not known. Our objective was to examine the role of c-Myb in the proliferation and differentiation of Sca1(+) adventitial VSMC progenitor cells. APPROACH AND RESULTS: Using mice with wild-type or hypomorphic c-myb (c-myb(h/h)), BrdU (bromodeoxyuridine) uptake and flow cytometry revealed defective proliferation of Sca1(+) adventitial VSMC progenitor cells at 8, 14, and 28 days post carotid artery denudation injury in c-myb(h/h) arteries. c-myb(h/h) cKit(+)CD34(-)Flk1(-)Sca1(+)CD45(-)Lin(-) cells failed to proliferate, suggesting that c-myb regulates the activation of specific Sca1(+) progenitor cells in vivo and in vitro. Although expression levels of transforming growth factor-ß1 did not vary between wild-type and c-myb(h/h) carotid arteries, in vitro differentiation of c-myb(h/h) Sca1(+) cells manifested defective transforming growth factor-ß1-induced VSMC differentiation. This is mediated by reduced transcriptional activation of myocardin because chromatin immunoprecipitation revealed c-Myb binding to the myocardin promoter only during differentiation of Sca1(+) cells, myocardin promoter mutagenesis identified 2 specific c-Myb-responsive binding sites, and adenovirus-mediated expression of myocardin rescued the phenotype of c-myb(h/h) progenitors. CONCLUSIONS: These data support a role for c-Myb in the regulation of VSMC progenitor cells and provide novel insight into how c-myb regulates VSMC differentiation through myocardin.
Assuntos
Túnica Adventícia/metabolismo , Ataxina-1/metabolismo , Lesões das Artérias Carótidas/metabolismo , Diferenciação Celular , Proliferação de Células , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Células-Tronco/metabolismo , Transativadores/metabolismo , Ativação Transcricional , Túnica Adventícia/efeitos dos fármacos , Túnica Adventícia/lesões , Túnica Adventícia/patologia , Animais , Sítios de Ligação , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , Artéria Carótida Primitiva/metabolismo , Artéria Carótida Primitiva/patologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Genótipo , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/lesões , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Proteínas Nucleares/genética , Fenótipo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myb/genética , Transdução de Sinais , Células-Tronco/efeitos dos fármacos , Células-Tronco/patologia , Fatores de Tempo , Transativadores/genética , Transcrição Gênica , Transfecção , Fator de Crescimento Transformador beta1/farmacologia , Remodelação VascularRESUMO
Expression of thioredoxin-interacting protein (TxNIP), an endogenous inhibitor of the thiol oxidoreductase thioredoxin, is augmented by high glucose (HG) and promotes oxidative stress. We previously reported that TxNIP-deficient mesangial cells showed protection from HG-induced reactive oxygen species, mitogen-activated protein kinase phosphorylation, and collagen expression. Here, we investigated the potential role of TxNIP in the pathogenesis of diabetic nephropathy (DN) in vivo. Wild-type (WT) control, TxNIP(-/-), and TxNIP(+/-) mice were rendered equally diabetic with low-dose streptozotocin. In contrast to effects in WT mice, diabetes did not increase albuminuria, proteinuria, serum cystatin C, or serum creatinine levels in TxNIP(-/-) mice. Whereas morphometric studies of kidneys revealed a thickened glomerular basement membrane and effaced podocytes in the diabetic WT mice, these changes were absent in the diabetic TxNIP(-/-) mice. Immunohistochemical analysis revealed significant increases in the levels of glomerular TGF-ß1, collagen IV, and fibrosis only in WT diabetic mice. Additionally, only WT diabetic mice showed significant increases in oxidative stress (nitrotyrosine, urinary 8-hydroxy-2-deoxy-guanosine) and inflammation (IL-1ß mRNA, F4/80 immunohistochemistry). Expression levels of Nox4-encoded mRNA and protein increased only in the diabetic WT animals. A significant loss of podocytes, assessed by Wilms' tumor 1 and nephrin staining and urinary nephrin concentration, was found in diabetic WT but not TxNIP(-/-) mice. Furthermore, in cultured human podocytes exposed to HG, TxNIP knockdown with siRNA abolished the increased mitochondrial O2 (-) generation and apoptosis. These data indicate that TxNIP has a critical role in the progression of DN and may be a promising therapeutic target.
Assuntos
Proteínas de Transporte/metabolismo , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/metabolismo , Tiorredoxinas/metabolismo , Albuminúria/etiologia , Animais , Antígenos de Diferenciação/análise , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas de Transporte/genética , Células Cultivadas , Colágeno Tipo IV/análise , Creatinina/sangue , Cistatina C/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Fibrose , Membrana Basal Glomerular/patologia , Glucose/farmacologia , Interleucina-1beta/genética , Glomérulos Renais/química , Glomérulos Renais/patologia , Proteínas de Membrana/análise , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , NADPH Oxidase 4 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Estresse Oxidativo , Oxigênio/metabolismo , Podócitos/química , Podócitos/efeitos dos fármacos , Podócitos/patologia , RNA Mensageiro/análise , Proteínas Repressoras/análise , Estreptozocina , Tiorredoxinas/genética , Fator de Crescimento Transformador beta1/análise , Proteínas WT1RESUMO
BACKGROUND: We previously showed that growth differentiation factor 5 (GDF5) limits infarct expansion post-myocardial infarction (MI). We now examine the acute post-MI role of GDF5 in cardiac rupture. METHODS AND RESULTS: Following permanent ligation of the left anterior descending artery, GDF5 deficiency (i.e., GDF5 knockout mice) reduced the incidence of cardiac rupture (4/24 vs. 17/24; P < .05), and improved survival over 28-d compared to wild-type (WT) mice (79% vs. 25%; P < .0001). Moreover, at 3-d post-MI, GDF5-deficient mice manifest: (a) reduced heart weight/body weight ratio (P < .0001) without differences in infarct size or cardiomyocyte size; (b) increased infarct zone expression of Col1a1 (P < .05) and Col3a1 (P < .01), suggesting increased myocardial fibrosis; and (c) reduced aortic and left ventricular peak systolic pressures (P ≤ .05), suggesting reduced afterload. Despite dysregulated inflammatory markers and reduced circulating monocytes in GDF5-deficient mice at 3-d post-MI, reciprocal bone marrow transplantation (BMT) failed to implicate GDF5 in BM-derived cells, suggesting the involvement of tissue-resident GDF5 expression in cardiac rupture. CONCLUSIONS: Loss of GDF5 reduces cardiac rupture post-MI with increased myocardial fibrosis and lower afterload, albeit at the cost of chronic adverse remodeling.
Assuntos
Fator 5 de Diferenciação de Crescimento , Ruptura Cardíaca , Infarto do Miocárdio , Animais , Camundongos , Modelos Animais de Doenças , Fibrose , Fator 5 de Diferenciação de Crescimento/genética , Fator 5 de Diferenciação de Crescimento/metabolismo , Ruptura Cardíaca/genética , Ruptura Cardíaca/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/complicações , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Miocárdio/patologiaRESUMO
Mechanisms mediating the cardioprotective actions of glucagon-like peptide 1 (GLP-1) were unknown. Here, we show in both ex vivo and in vivo models of ischemic injury that treatment with GLP-1(28-36), a neutral endopeptidase-generated (NEP-generated) metabolite of GLP-1, was as cardioprotective as GLP-1 and was abolished by scrambling its amino acid sequence. GLP-1(28-36) enters human coronary artery endothelial cells (caECs) through macropinocytosis and acts directly on mouse and human coronary artery smooth muscle cells (caSMCs) and caECs, resulting in soluble adenylyl cyclase Adcy10-dependent (sAC-dependent) increases in cAMP, activation of protein kinase A, and cytoprotection from oxidative injury. GLP-1(28-36) modulates sAC by increasing intracellular ATP levels, with accompanying cAMP accumulation lost in sAC-/- cells. We identify mitochondrial trifunctional protein-α (MTPα) as a binding partner of GLP-1(28-36) and demonstrate that the ability of GLP-1(28-36) to shift substrate utilization from oxygen-consuming fatty acid metabolism toward oxygen-sparing glycolysis and glucose oxidation and to increase cAMP levels is dependent on MTPα. NEP inhibition with sacubitril blunted the ability of GLP-1 to increase cAMP levels in coronary vascular cells in vitro. GLP-1(28-36) is a small peptide that targets novel molecular (MTPα and sAC) and cellular (caSMC and caEC) mechanisms in myocardial ischemic injury.
Assuntos
Cardiotônicos/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Mitocôndrias Cardíacas/metabolismo , Subunidade alfa da Proteína Mitocondrial Trifuncional/metabolismo , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/prevenção & controle , Animais , Vasos Coronários/metabolismo , Vasos Coronários/patologia , AMP Cíclico/genética , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Peptídeo 1 Semelhante ao Glucagon/genética , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/patologia , Subunidade alfa da Proteína Mitocondrial Trifuncional/genética , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Isquemia Miocárdica/genética , Isquemia Miocárdica/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Sistemas do Segundo Mensageiro/genéticaRESUMO
The proto-oncogene c-myb (and corresponding nuclear transcription factor, c-Myb) regulates the proliferation and differentiation of hematologic and vascular smooth muscle cells; however, the role of c-Myb in blood pressure regulation is unknown. Here, we show that mice homozygous for a hypomorphic c-myb allele ( c-myb h/h) conferring reduced c-Myb activity manifest reduced peripheral blood and kidney B220+ B-cells and have decreased systolic (104±2 versus 120±1 mm Hg; P<0.0001) and diastolic blood pressure (71±2 versus 83±1 mm Hg; P<0.0001) compared with WT (wild type) mice. Additionally, c-myb h/h mice had lower susceptibility to deoxycorticosterone acetate-salt experimental hypertension. Although cardiac (echocardiography) and resistance artery (perfusion myography) functions were normal, metabolic cage studies revealed that c-myb h/h mice had increased 24-hour urine output and sodium excretion versus WT. Reconstitution of WT mice with c-myb h/h bone marrow transplant and chimeric bone marrow transplant using mice lacking B-cells ( J H T; h/h>WT and h/h:J H T>WT, respectively) decreased blood pressure and increased 24-hour urine output compared with controls ( WT>WT; WT:J H T>WT). J H T mice also had decreased systolic (103±2 versus 115±1 mm Hg; P<0.0001) and diastolic blood pressure (71±2 versus 79±1; P<0.01) and increased 24-hour urine output versus WT. Real-time quantitative reverse transcription polymerase chain reaction of kidney medulla revealed reduced V2R (vasopressin receptor 2) expression in c-myb h/h and J H T mice. These data implicate B-cells in the regulation of V2R and its associated effects on salt and water handling and blood pressure homeostasis.
Assuntos
Linfócitos B/metabolismo , Pressão Sanguínea/fisiologia , Hipertensão/imunologia , Miócitos de Músculo Liso/metabolismo , Animais , Linfócitos B/patologia , Diferenciação Celular , Modelos Animais de Doenças , Regulação da Expressão Gênica , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/patologia , Proteínas Proto-Oncogênicas c-myb/biossíntese , Proteínas Proto-Oncogênicas c-myb/genética , RNA/genéticaRESUMO
Mechanisms that govern transcriptional regulation of inflammation in atherosclerosis remain largely unknown. Here, we identify the nuclear transcription factor c-Myb as an important mediator of atherosclerotic disease in mice. Atherosclerosis-prone animals fed a diet high in cholesterol exhibit increased levels of c-Myb in the bone marrow. Use of mice that either harbor a c-Myb hypomorphic allele or where c-Myb has been preferentially deleted in B cell lineages revealed that c-Myb potentiates atherosclerosis directly through its effects on B lymphocytes. Reduced c-Myb activity prevents the expansion of atherogenic B2 cells yet associates with increased numbers of IgM-producing antibody-secreting cells (IgM-ASCs) and elevated levels of atheroprotective oxidized low-density lipoprotein (OxLDL)-specific IgM antibodies. Transcriptional profiling revealed that c-Myb has a limited effect on B cell function but is integral in maintaining B cell progenitor populations in the bone marrow. Thus, targeted disruption of c-Myb beneficially modulates the complex biology of B cells in cardiovascular disease.
Assuntos
Células Produtoras de Anticorpos/imunologia , Aterosclerose/genética , Aterosclerose/imunologia , Imunoglobulina M/metabolismo , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/imunologia , Animais , Células Produtoras de Anticorpos/metabolismo , Aterosclerose/patologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Genes myb , Masculino , CamundongosRESUMO
BACKGROUND: MicroRNAs (miR) are small non-coding RNAs that regulate diverse biological functions. The bicistronic gene miR-143/145 determines cell fate and phenotype of vascular smooth muscle cells (VSMC), in part, by destabilizing Elk-1 mRNA. The transcription factor c-Myb also regulates differentiation and proliferation of VSMC, and here we test whether these effects may be mediated by miR-143/145. METHODS & RESULTS: Flow cytometry of cardiovascular-directed d3.75 embryoid bodies (EBs) isolated smooth muscle progenitors with specific cell surface markers. In c-myb knockout (c-myb -/-) EB, these progenitors manifest low levels of miR-143 (19%; p<0.05) and miR-145 (6%; p<0.01) expression as compared to wild-type (wt) EB. Primary VSMC isolated from transgenic mice with diminished expression (c-myblx/lx) or reduced activity (c-mybh/h) of c-Myb also manifest low levels of miR-143 (c-myblx/lx: 50%; c-mybh/h: 41%), and miR-145 (c-myblx/lx: 49%; c-mybh/h: 56%), as compared to wt (P<0.05). Sequence alignment identified four putative c-Myb binding sites (MBS1-4) in the proximal promoter (PP) of the miR-143/145 gene. PP-reporter constructs revealed that point mutations in MBS1 and MBS4 abrogated c-Myb-dependent transcription from the miR-143/145 PP (P<0.01). Chromatin immunoprecipitation (ChIP) revealed preferential c-Myb binding at MBS4 (p<0.001). By conjugating Elk-1 3'-untranslated region (UTR) to a reporter and co-transducing wt VSMC with this plus a miR-143-antagomir, and co-transducing c-myblx/lx VSMC with this plus a miR-143-mimic, we demonstrate that c-Myb's ability to repress Elk-1 is mediated by miR-143. CONCLUSION: c-Myb regulates VSMC gene expression by transcriptional activation of miR-143/145.
Assuntos
MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Regiões 3' não Traduzidas , Animais , Antagomirs/metabolismo , Sítios de Ligação , Células Cultivadas , Imunoprecipitação da Cromatina , Camundongos , Camundongos Knockout , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Mutação Puntual , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myb/genética , Ativação Transcricional/genética , Proteínas Elk-1 do Domínio ets/genética , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
AIMS: Despite the diverse developmental origins of vascular smooth muscle cells (VSMCs), recent attempts to generate VSMCs from human embryonic stem cells (hESCs) differentiated along various lineages did not yield distinct cell phenotypes. The aim of this study was to derive and characterize functional coronary-like VSMCs from hESCs using serum-free cardiac-directed differentiation. METHODS AND RESULTS: Embryoid bodies (EBs) from three pluripotent stem cell lines subjected to cardiac-directed differentiation in defined media were characterized over 30 days for VSMC-specific gene expression by qRT-PCR, immunofluorescence microscopy and fluorescence-activated cell sorting (FACS). EBs composed of cardiomyocytes, endothelial cells (ECs), fibroblasts, and VSMCs underwent FACS on d28 to reveal that the VSMCs form a distinct subpopulation, which migrate with ECs in an in vitro angiogenesis assay. To enrich for VSMCs, d28 EBs were dissociated and cultured as monolayers. Over several passages, mRNA and protein levels of cardiomyocyte, endothelial, and fibroblast markers were abolished, whereas those of mature VSMCs were unchanged. Vascular endothelial growth factor and basic fibroblast growth factor were critical for the separation of the cardiac and VSMC lineages in EBs, and for the enrichment of functional VSMCs in monolayer cultures. Calcium cycling and cell shortening responses to vasoconstrictors in hESC-derived VSMCs in vitro were indistinguishable from primary human coronary artery SMCs, and distinct from bladder and aorta SMCs. VSMCs identically derived from green fluorescent protein -expressing hESCs integrated in and contributed to new vessel formation in vivo. CONCLUSION: The ability to generate hESC-derived functional human coronary-like VSMCs in serum-free conditions has implications for disease modelling, drug screening, and regenerative therapies.
Assuntos
Diferenciação Celular , Vasos Coronários/citologia , Células-Tronco Embrionárias/citologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Actinas/análise , Animais , Cálcio/metabolismo , Células Cultivadas , Meios de Cultura Livres de Soro , Fator 2 de Crescimento de Fibroblastos/fisiologia , Humanos , Camundongos , Camundongos SCID , Neovascularização Fisiológica , RNA Mensageiro/análise , Fator A de Crescimento do Endotélio Vascular/fisiologia , Vasoconstrição/efeitos dos fármacosRESUMO
Chronic exposure to high glucose leads to diabetic nephropathy characterized by increased mesangial matrix protein (e.g., collagen) accumulation. Altered cell signaling and gene expression accompanied by oxidative stress have been documented. The contribution of the tyrosine kinase, c-Src (Src), which is sensitive to oxidative stress, was examined. Cultured rat mesangial cells were exposed to high glucose (25 mmol/L) in the presence and absence of Src inhibitors (PP2, SU6656), Src small interfering RNA (siRNA), and the tumor necrosis factor-α-converting enzyme (TACE) inhibitor, TAPI-2. Src was investigated in vivo by administration of PP2 to streptozotocin (STZ)-induced diabetic DBA2/J mice. High glucose stimulated Src, TACE, epidermal growth factor receptor (EGFR), mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK1/2, p38), and collagen IV accumulation in mesangial cells. PP2 and SU6656 blocked high glucose-stimulated phosphorylation of Src Tyr-416, EGFR, and MAPKs. These inhibitors and Src knockdown by siRNA, as well as TAPI-2, also abrogated high glucose-induced phosphorylation of these targets and collagen IV accumulation. In STZ-diabetic mice, albuminuria, increased Src pTyr-416, TACE activation, ERK and EGFR phosphorylation, glomerular collagen accumulation, and podocyte loss were inhibited by PP2. These data indicate a role for Src in a high glucose-Src-TACE-heparin-binding epidermal growth factor-EGFR-MAPK-signaling pathway to collagen accumulation. Thus, Src may provide a novel therapeutic target for diabetic nephropathy.
Assuntos
Proteínas ADAM/metabolismo , Colágeno Tipo IV/biossíntese , Nefropatias Diabéticas/prevenção & controle , Receptores ErbB/metabolismo , Quinases da Família src/antagonistas & inibidores , Proteína ADAM17 , Albuminúria/prevenção & controle , Animais , Diabetes Mellitus Experimental/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glucose/administração & dosagem , Masculino , Células Mesangiais/metabolismo , Camundongos , Podócitos/efeitos dos fármacos , Podócitos/fisiologia , Pirimidinas/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacosRESUMO
During the inflammatory response that drives atherogenesis, macrophages accumulate progressively in the expanding arterial wall. The observation that circulating monocytes give rise to lesional macrophages has reinforced the concept that monocyte infiltration dictates macrophage buildup. Recent work has indicated, however, that macrophage accumulation does not depend on monocyte recruitment in some inflammatory contexts. We therefore revisited the mechanism underlying macrophage accumulation in atherosclerosis. In murine atherosclerotic lesions, we found that macrophages turn over rapidly, after 4 weeks. Replenishment of macrophages in these experimental atheromata depends predominantly on local macrophage proliferation rather than monocyte influx. The microenvironment orchestrates macrophage proliferation through the involvement of scavenger receptor A (SR-A). Our study reveals macrophage proliferation as a key event in atherosclerosis and identifies macrophage self-renewal as a therapeutic target for cardiovascular disease.
Assuntos
Aterosclerose/imunologia , Aterosclerose/patologia , Proliferação de Células , Macrófagos/fisiologia , Animais , Células Cultivadas , Inflamação , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/fisiologiaRESUMO
The angiostatic nature of pharmacological doses of glucocorticoid steroids is well known. However, the consequences of pathophysiological elevation of endogenous glucocorticoids are not well established. In the current study, we hypothesized that the angiostatic effect of corticosterone, an endogenous glucocorticoid in rodents, occurs through multi-faceted alterations in skeletal muscle microvascular endothelial cell proliferation, migration, and proteolysis. Chronic corticosterone treatment significantly reduced the capillary to fiber ratio in the tibialis anterior muscle compared to that of placebo-treated rats. Corticosterone inhibited endothelial cell sprouting from capillary segments ex vivo. Similarly, 3-dimensional endothelial cell spheroids treated with corticosterone for 48 hours showed evidence of sprout regression and reduced sprout length. Endothelial cell proliferation was reduced in corticosterone treated cells, coinciding with elevated FoxO1 and reduced VEGF production. Corticosterone treated endothelial cells exhibited reduced migration, which correlated with a reduction in RhoA activity. Furthermore, corticosterone treated endothelial cells in both 3-dimensional and monolayer cultures had decreased MMP-2 production and activation resulting in decreased proteolysis by endothelial cells, limiting their angiogenic potential. Promoter assays revealed that corticosterone treatment transcriptionally repressed MMP-2, which may map to a predicted GRE between -1510 and -1386 bp of the MMP-2 promoter. Additionally, Sp1, a known transcriptional activator of MMP-2 was decreased following corticosterone treatment. This study provides new insights into the mechanisms by which pathophysiological levels of endogenous glucocorticoids may exert angiostatic effects.