RESUMEN
OBJECTIVE: Vascular lineage differentiation of stem/progenitor cells can contribute to both tissue repair and exacerbation of vascular diseases such as in vein grafts. The role of macrophages in controlling vascular progenitor differentiation is largely unknown and may play an important role in graft development. This study aims to identify the role of macrophages in vascular stem/progenitor cell differentiation and thereafter elucidate the mechanisms that are involved in the macrophage- mediated process. APPROACH AND RESULTS: We provide in vitro evidence that macrophages can induce endothelial cell (EC) differentiation of the stem/progenitor cells while simultaneously inhibiting their smooth muscle cell differentiation. Mechanistically, both effects were mediated by macrophage-derived tumor necrosis factor-α (TNF-α) via TNF-α receptor 1 and canonical nuclear factor-κB activation. Although the overexpression of p65 enhanced EC (or attenuated smooth muscle cell) differentiation, p65 or TNF-α receptor 1 knockdown using lentiviral short hairpin RNA inhibited EC (or rescued smooth muscle cell) differentiation in response to TNF-α. Furthermore, TNF-α-mediated EC differentiation was driven by direct binding of nuclear factor-κB (p65) to specific VE-cadherin promoter sequences. Subsequent experiments using an ex vivo decellularized vessel scaffold confirmed an increase in the number of ECs and reduction in smooth muscle cell marker expression in the presence of TNF-α. The lack of TNF-α in a knockout mouse model of vein graft decreased endothelialization and significantly increased thrombosis formation. CONCLUSIONS: Our study highlights the role of macrophages in directing vascular stem/progenitor cell lineage commitment through TNF-α-mediated TNF-α receptor 1 and nuclear factor-κB activation that is likely required for endothelial repair in vascular diseases such as vein graft.
Asunto(s)
Células Madre Adultas/citología , Células Endoteliales/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/fisiología , Macrófagos Peritoneales/fisiología , Miocitos del Músculo Liso/efectos de los fármacos , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Células Madre Adultas/efectos de los fármacos , Proteínas Angiogénicas/farmacología , Animales , Antígenos CD/biosíntesis , Antígenos CD/genética , Apoptosis , Cadherinas/biosíntesis , Cadherinas/genética , Línea Celular , Linaje de la Célula , Medios de Cultivo Condicionados/farmacología , Células Endoteliales/citología , Endotelio Vascular/citología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Interleucina-6/farmacología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Neointima/patología , Neovascularización Fisiológica/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/fisiología , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Quimera por Radiación , Receptores Tipo I de Factores de Necrosis Tumoral/efectos de los fármacos , Receptores Tipo I de Factores de Necrosis Tumoral/fisiología , Proteínas Recombinantes/farmacología , Transducción de Señal , Trombofilia/etiología , Trombofilia/fisiopatología , Andamios del Tejido , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/deficiencia , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/farmacología , Venas/trasplanteRESUMEN
OBJECTIVE: Sirolimus-eluting stent therapy has achieved considerable success in overcoming coronary artery restenosis. However, there remain a large number of patients presenting with restenosis after the treatment, and the source of its persistence remains unclarified. Although recent evidence supports the contribution of vascular stem/progenitor cells in restenosis formation, their functional and molecular responses to sirolimus are largely unknown. APPROACH AND RESULTS: Using an established technique, vascular progenitor cells were isolated from adventitial tissues of mouse vessel grafts and purified with microbeads specific for stem cell antigen-1. We provide evidence that vascular progenitor cells treated with sirolimus resulted in an induction of their migration in both transwell and wound healing models, clearly mediated by CXCR4 activation. We confirmed the sirolimus-mediated increase of migration from the adventitial into the intima side using an ex vivo decellularized vessel scaffold, where they form neointima-like lesions that expressed high levels of smooth muscle cell (SMC) markers (SM-22α and calponin). Subsequent in vitro studies confirmed that sirolimus can induce SMC but not endothelial cell differentiation of progenitor cells. Mechanistically, we showed that sirolimus-induced progenitor-SMC differentiation was mediated via epidermal growth factor receptor and extracellular signal-regulated kinase 1/2 activation that lead to ß-catenin nuclear translocation. The ablation of epidermal growth factor receptor, extracellular signal-regulated kinase 1/2, or ß-catenin attenuated sirolimus-induced SM-22α promoter activation and SMC differentiation. CONCLUSIONS: These findings provide direct evidence of sirolimus-induced progenitor cell migration and differentiation into SMC via CXCR4 and epidermal growth factor receptor/extracellular signal-regulated kinase/ß-catenin signal pathways, thus implicating a novel mechanism of restenosis formation after sirolimus-eluting stent treatment.
Asunto(s)
Células Madre Adultas/efectos de los fármacos , Adventicia/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Sirolimus/farmacología , beta Catenina/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Células Madre Adultas/enzimología , Adventicia/citología , Adventicia/enzimología , Animales , Antígenos Ly/metabolismo , Biomarcadores/metabolismo , Proteínas de Unión al Calcio/metabolismo , Células Cultivadas , Constricción Patológica , Activación Enzimática , Proteínas de la Membrana/metabolismo , Ratones , Proteínas de Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Miocitos del Músculo Liso/enzimología , Interferencia de ARN , Receptores CXCR4/agonistas , Receptores CXCR4/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Andamios del Tejido , Transfección , beta Catenina/genética , CalponinasRESUMEN
OBJECTIVE: This study was designed to carry out the characterization of stem cells within the adventitia and to elucidate their functional role in the pathogenesis of vein graft atherosclerosis. APPROACH AND RESULTS: A mouse vein graft model was used to investigate the functional role of adventitial stem/progenitor cells on atherosclerosis. The adventitia of vein grafts underwent significant remodeling during early stages of vessel grafting and displayed markedly heterogeneous cell compositions. Immunofluorescence staining indicated a significant number of stem cell antigen-1-positive cells that were closely located to vasa vasorum. In vitro clonogenic assays demonstrated 1% to 11% of growing rates from adventitial cell cultures, most of which could be differentiated into smooth muscle cells (SMCs). These stem cell antigen-1-positive cells also displayed a potential to differentiate into adipogenic, osteogenic, or chondrogenic lineages in vitro. In light of the proatherogenic roles of SMCs in atherosclerosis, we focused on the functional roles of progenitor-SMC differentiation, in which we subsequently demonstrated that it was driven by direct interaction of the integrin/collagen IV axis. The ex vivo bioreactor system revealed the migratory capacity of stem cell antigen-1-positive progenitor cells into the vessel wall in response to stromal cell-derived factor-1. Stem cell antigen-1-positive cells that were applied to the outer layer of vein grafts showed enhanced atherosclerosis in apolipoprotein E-deficient mice, which contributed to ≈ 30% of neointimal SMCs. CONCLUSIONS: We demonstrate that during pathological conditions in vein grafting, the adventitia harbors stem/progenitor cells that can actively participate in the pathogenesis of vascular disease via differentiation into SMCs.
Asunto(s)
Aterosclerosis/patología , Linaje de la Célula/fisiología , Oclusión de Injerto Vascular/patología , Neointima/patología , Células Madre/patología , Venas/trasplante , Adventicia/patología , Animales , Antígenos Ly/metabolismo , Apolipoproteínas E/genética , Diferenciación Celular/fisiología , Células Cultivadas , Colágeno Tipo IV/metabolismo , Modelos Animales de Enfermedad , Integrinas/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Células Madre/metabolismo , Trasplante Autólogo , Venas/patologíaRESUMEN
OBJECTIVE: Smooth muscle cell (SMC) differentiation is a critical process during cardiovascular formation and development, but the underlying molecular mechanism remains unclear. METHODS AND RESULTS: Here we demonstrated that chromobox protein homolog 3 (Cbx3) is crucial for SMC differentiation from stem cells and that the chromodomain and chromoshadow domain of Cbx3 are responsible for Cbx3-induced SMC differentiation. Moreover, we identified that 4 amino acids (165 to 168) within the chromoshadow domain of Cbx3 are key elements for Cbx3 interaction with Dia-1- and Cbx3-induced SMC differentiation. Mechanistically, we found that Cbx3 mediates SMC differentiation through modulating serum response factor (SRF) recruitment to the promoters of SMC genes, in which the interaction between Cbx3 and Dia-1/SRF plays a crucial role in this process. Moreover, our in vivo study demonstrated that the misexpression of Cbx3 within neural crest cells of chick embryos resulted in the death of chick embryos at early stages because of the maldevelopment of branchial arch arteries. CONCLUSIONS: Our findings suggest that the interaction between Cbx3 and Dia-1/SRF is essential for SMC differentiation from stem cells and for the development of functional cardiovascular system.
Asunto(s)
Proteínas Aviares/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Proteínas Aviares/genética , Región Branquial/embriología , Región Branquial/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Embrión de Pollo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Forminas , Técnicas de Silenciamiento del Gen , Ratones , Neovascularización Fisiológica/genética , Cresta Neural/embriología , Cresta Neural/metabolismo , Regiones Promotoras Genéticas , Dominios y Motivos de Interacción de Proteínas , Factor de Respuesta Sérica/metabolismo , Proteínas ViralesRESUMEN
NADPH oxidase (Nox4) produces reactive oxygen species (ROS) that are important for vascular smooth muscle cell (SMC) behavior, but the potential impact of Nox4 in stem cell differentiation is unknown. When mouse embryonic stem (ES) cells were plated on collagen IV-coated dishes/flasks, a panel of SMC-specific genes was significantly and consistently upregulated. Nox4 expression was markedly correlated with such a gene induction as confirmed by real-time PCR, immunofluorescence, and Western blot analysis. Overexpression of Nox4 specifically resulted in increased SMC marker production, whereas knockdown of Nox4 induced a decrease. Furthermore, SMC-specific transcription factors, including serum response factor (SRF) and myocardin were activated by Nox4 gene expression. Moreover, Nox4 was demonstrated to drive SMC differentiation through generation of H(2)O(2). Confocal microscopy analysis indicates that SRF was translocated into the nucleus during SMC differentiation in which SRF was phosphorylated. Additionally, autosecreted transforming growth factor (TGF)-beta(1) activated Nox4 and promoted SMC differentiation. Interestingly, cell lines generated from stem cells by Nox4 transfection and G418 selection displayed a characteristic of mature SMCs, including expression of SMC markers and cells with contractile function. Thus we demonstrate for the first time that Nox4 is crucial for SMC differentiation from ES cells, and enforced Nox4 expression can maintain differentiation status and functional features of stem cell-derived SMCs, highlighting its impact on vessel formation in vivo and vascular tissue engineering in the future.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/enzimología , Peróxido de Hidrógeno/metabolismo , Miocitos del Músculo Liso/enzimología , NADPH Oxidasas/metabolismo , Animales , Comunicación Autocrina , Diferenciación Celular/genética , Línea Celular , Colágeno Tipo IV/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Ratones , NADPH Oxidasa 4 , NADPH Oxidasas/genética , Proteínas Nucleares/metabolismo , Fenotipo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Respuesta Sérica/metabolismo , Factores de Tiempo , Transactivadores/metabolismo , Transfección , Factor de Crecimiento Transformador beta1/metabolismo , Regulación hacia ArribaRESUMEN
Inflammation and TNF-alpha signaling play a central role in most of the pathological conditions where cell transplantation could be applied. As shown by initial experiments, embryonic stem (ES) cells and ES-cell derived vascular cells express very low levels of TNF-alpha receptor I (TNFRp55) and thus do not induce cytokine expression in response to TNF-alpha stimulation. Transient transfection analysis of wild-type or deletion variants of the TNFRp55 gene promoter showed a strong activity for a 250-bp fragment in the upstream region of the gene. This activity was abolished by mutations targeting the Sp1/Sp3 or AP1 binding sites. Moreover, treatment with trichostatin A (TSA) led to a pronounced increase in TNFRp55 mRNA and promoter activity. Overexpression of Sp1 or c-fos further enhanced the TSA-induced luciferase activity, and this response was attenuated by Sp3 or c-jun coexpression. Additional experiments revealed that TSA did not affect the Sp1/Sp3 ratio but caused transcriptional activation of the c-fos gene. Thus, we provide the first evidence that ES and ES-cell-derived vascular cells lack cytokine expression in response to TNF-alpha stimulation due to low levels of c-fos and transcriptional activation of Sp1 that can be regulated by inhibition of histone deacetylase activity.