RESUMEN
Chromatin priming promotes cell-type-specific gene expression, lineage differentiation, and development. The mechanism of chromatin priming has not been fully understood. Here, we report that mouse hematopoietic stem and progenitor cells (HSPCs) lacking the Baf155 subunit of the BAF (BRG1/BRM-associated factor) chromatin remodeling complex produce a significantly reduced number of mature blood cells, leading to a failure of hematopoietic regeneration upon transplantation and 5-fluorouracil (5-FU) injury. Baf155-deficient HSPCs generate particularly fewer neutrophils, B cells, and CD8+ T cells at homeostasis, supporting a more immune-suppressive tumor microenvironment and enhanced tumor growth. Single-nucleus multiomics analysis reveals that Baf155-deficient HSPCs fail to establish accessible chromatin in selected regions that are enriched for putative enhancers and binding motifs of hematopoietic lineage transcription factors. Our study provides a fundamental mechanistic understanding of the role of Baf155 in hematopoietic lineage chromatin priming and the functional consequences of Baf155 deficiency in regeneration and tumor immunity.
Asunto(s)
Diferenciación Celular , Cromatina , Hematopoyesis , Células Madre Hematopoyéticas , Animales , Cromatina/metabolismo , Ratones , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/citología , Ratones Endogámicos C57BL , Regeneración , Fluorouracilo/farmacología , Factores de Transcripción/metabolismo , Factores de Transcripción/genéticaRESUMEN
Tumor angiogenesis and immunity show an inverse correlation in cancer progression and outcome1. Here, we report that ZBTB46, a repressive transcription factor and a widely accepted marker for classical dendritic cells (DCs)2,3, controls both tumor angiogenesis and immunity. Zbtb46 was downregulated in both DCs and endothelial cells by tumor-derived factors to facilitate robust tumor growth. Zbtb46 downregulation led to a hallmark pro-tumor microenvironment (TME), including dysfunctional vasculature and immunosuppressive conditions. Analysis of human cancer data revealed a similar association of low ZBTB46 expression with an immunosuppressive TME and a worse prognosis. In contrast, enforced Zbtb46 expression led to TME changes to restrict tumor growth. Mechanistically, Zbtb46-deficient endothelial cells were highly angiogenic, and Zbtb46-deficient bone marrow progenitors upregulated Cebpb and diverted the DC program to immunosuppressive myeloid lineage output, potentially explaining the myeloid lineage skewing phenomenon in cancer4. Conversely, enforced Zbtb46 expression normalized tumor vessels and, by suppressing Cebpb, skewed bone marrow precursors toward immunostimulatory myeloid lineage output, leading to an immune-hot TME. Remarkably, Zbtb46 mRNA treatment synergized with anti-PD1 immunotherapy to improve tumor management in preclinical models. These findings identify ZBTB46 as a critical factor for angiogenesis and for myeloid lineage skewing in cancer and suggest that maintaining its expression could have therapeutic benefits.
Asunto(s)
Células Dendríticas , Neovascularización Patológica , Microambiente Tumoral , Animales , Microambiente Tumoral/inmunología , Ratones , Neovascularización Patológica/inmunología , Neovascularización Patológica/genética , Humanos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Ratones Endogámicos C57BL , Neoplasias/inmunología , Neoplasias/patología , Neoplasias/genética , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/inmunología , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/genética , Femenino , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Ratones Noqueados , Angiogénesis , Factores de TranscripciónRESUMEN
Extramedullary hematopoiesis (EMH) expands hematopoietic capacity outside of the bone marrow in response to inflammatory conditions, including infections and cancer. Because of its inducible nature, EMH offers a unique opportunity to study the interaction between hematopoietic stem and progenitor cells (HSPCs) and their niche. In cancer patients, the spleen frequently serves as an EMH organ and provides myeloid cells that may worsen pathology. Here, we examined the relationship between HSPCs and their splenic niche in EMH in a mouse breast cancer model. We identify tumor produced IL-1α and leukemia inhibitory factor (LIF) acting on splenic HSPCs and splenic niche cells, respectively. IL-1α induced TNFα expression in splenic HSPCs, which then activated splenic niche activity, while LIF induced proliferation of splenic niche cells. IL-1α and LIF display cooperative effects in activating EMH and are both up-regulated in some human cancers. Together, these data expand avenues for developing niche-directed therapies and further exploring EMH accompanying inflammatory pathologies like cancer.
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Enfermedades Hematológicas , Hematopoyesis Extramedular , Neoplasias , Humanos , Animales , Ratones , Hematopoyesis Extramedular/fisiología , Factor Inhibidor de Leucemia/farmacología , Interleucina-1alfa/farmacología , HematopoyesisRESUMEN
The diversity of mononuclear phagocyte (MNP) subpopulations across tissues is one of the key physiological characteristics of the immune system. Here, we focus on understanding the metabolic variability of MNPs through metabolic network analysis applied to three large-scale transcriptional datasets: we introduce (1) an ImmGen MNP open-source dataset of 337 samples across 26 tissues; (2) a myeloid subset of ImmGen Phase I dataset (202 MNP samples); and (3) a myeloid mouse single-cell RNA sequencing (scRNA-seq) dataset (51,364 cells) assembled based on Tabula Muris Senis. To analyze such large-scale datasets, we develop a network-based computational approach, genes and metabolites (GAM) clustering, for unbiased identification of the key metabolic subnetworks based on transcriptional profiles. We define 9 metabolic subnetworks that encapsulate the metabolic differences within MNP from 38 different tissues. Obtained modules reveal that cholesterol synthesis appears particularly active within the migratory dendritic cells, while glutathione synthesis is essential for cysteinyl leukotriene production by peritoneal and lung macrophages.
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Fagocitos , Análisis de la Célula Individual , Animales , RatonesRESUMEN
Immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment. However, only a fraction of patients respond to ICB therapy. Accurate prediction of patients to likely respond to ICB would maximize the efficacy of ICB therapy. The tumor microenvironment (TME) dictates tumor progression and therapy outcome. Here, we classify the TME by analyzing the transcriptome from 11,069 cancer patients based on angiogenesis and T cell activity. We find three distinct angio-immune TME subtypes conserved across 30 non-hematological cancers. There is a clear inverse relationship between angiogenesis and anti-tumor immunity in TME. Remarkably, patients displaying TME with low angiogenesis with strong anti-tumor immunity show the most significant responses to ICB therapy in four cancer types. Re-evaluation of the renal cell carcinoma clinical trials provides compelling evidence that the baseline angio-immune state is robustly predictive of ICB responses. This study offers a rationale for incorporating baseline angio-immune scores for future ICB treatment strategies.
Asunto(s)
Carcinoma de Células Renales , Neoplasias Renales , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Microambiente Tumoral , Carcinoma de Células Renales/tratamiento farmacológico , Transcriptoma , Neoplasias Renales/tratamiento farmacológicoRESUMEN
Nuclear envelope membrane proteins (NEMPs) are a conserved family of nuclear envelope (NE) proteins that reside within the inner nuclear membrane (INM). Even though Nemp1 knockout (KO) mice are overtly normal, they display a pronounced splenomegaly. This phenotype and recent reports describing a requirement for NE openings during erythroblasts terminal maturation led us to examine a potential role for Nemp1 in erythropoiesis. Here, we report that Nemp1 KO mice show peripheral blood defects, anemia in neonates, ineffective erythropoiesis, splenomegaly, and stress erythropoiesis. The erythroid lineage of Nemp1 KO mice is overrepresented until the pronounced apoptosis of polychromatophilic erythroblasts. We show that NEMP1 localizes to the NE of erythroblasts and their progenitors. Mechanistically, we discovered that NEMP1 accumulates into aggregates that localize near or at the edge of NE openings and Nemp1 deficiency leads to a marked decrease of both NE openings and ensuing enucleation. Together, our results for the first time demonstrate that NEMP1 is essential for NE openings and erythropoietic maturation in vivo and provide the first mouse model of defective erythropoiesis directly linked to the loss of an INM protein.
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Membrana Nuclear , Esplenomegalia , Ratones , Animales , Eritroblastos/metabolismo , Núcleo Celular/metabolismo , Eritropoyesis/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones NoqueadosRESUMEN
The cross-talk between angiogenesis and immunity within the tumor microenvironment (TME) is critical for tumor prognosis. While pro-angiogenic and immunosuppressive TME promote tumor growth, anti-angiogenic and immune stimulatory TME inhibit tumor progression. Therefore, there is a great interest in achieving vascular normalization to improve drug delivery and enhance antitumor immunity. However, anti-vascular endothelial growth factor (VEGF) mechanisms to normalize tumor vessels have offered limited therapeutic efficacies for patients with cancer. Here, we report that Myct1, a direct target of ETV2, was nearly exclusively expressed in endothelial cells. In preclinical mouse tumor models, Myct1 deficiency reduced angiogenesis, enhanced high endothelial venule formation, and promoted antitumor immunity, leading to restricted tumor progression. Analysis of The Cancer Genome Atlas (TCGA) datasets revealed a significant (P < 0.05) correlation between MYCT1 expression, angiogenesis, and antitumor immunity in human cancers, as suggested by decreased FOXP3 expression and increased antitumor macrophages in patients with low MYCT1 expression. Mechanistically, MYCT1 interacted with tight junction protein Zona Occludens 1 and regulated Rho GTPase-mediated actin cytoskeleton dynamics, thereby promoting endothelial motility in the angiogenic environment. Myct1-deficient endothelial cells facilitated trans-endothelial migration of cytotoxic T lymphocytes and polarization of M1 macrophages. Myct1 targeting combined with anti-PD1 treatment significantly (P < 0.05) increased complete tumor regression and long-term survival in anti-PD1-responsive and -refractory tumor models in mice. Our data collectively support a critical role for Myct1 in controlling tumor angiogenesis and reprogramming tumor immunity. Myct1-targeted vascular control, in combination with immunotherapy, may become an exciting therapeutic strategy.
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Células Endoteliales , Neovascularización Patológica , Microambiente Tumoral , Animales , Línea Celular Tumoral , Humanos , Inmunoterapia , Ratones , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Proteínas Nucleares , Factores de TranscripciónRESUMEN
The mammalian SWitch/Sucrose Non-Fermentable (SWI/SNF) chromatin-remodeling BAF (BRG1/BRM-associated factor) complex plays an essential role in developmental and pathological processes. We show that the deletion of Baf155, which encodes a subunit of the BAF complex, in the Tie2(+) lineage (Baf155 (CKO) leads to defects in yolk sac myeloid and definitive erythroid (EryD) lineage differentiation from erythromyeloid progenitors (EMPs). The chromatin of myeloid gene loci in Baf155 CKO EMPs is mostly inaccessible and enriched mainly by the ETS binding motif. BAF155 interacts with PU.1 and is recruited to PU.1 target gene loci together with p300 and KDM6a. Treatment of Baf155 CKO embryos with GSK126, an H3K27me2/3 methyltransferase EZH2 inhibitor, rescues myeloid lineage gene expression. This study uncovers indispensable BAF-mediated chromatin remodeling of myeloid gene loci at the EMP stage. Future studies exploiting epigenetics in the generation and application of EMP derivatives for tissue repair, regeneration, and disease are warranted.
Asunto(s)
Linaje de la Célula/fisiología , Ensamble y Desensamble de Cromatina/fisiología , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular/fisiología , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/genética , Proteínas Cromosómicas no Histona/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Epigénesis Genética/genética , Células Eritroides/metabolismo , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Células Madre Embrionarias de Ratones/metabolismo , Células Mieloides/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismoRESUMEN
Human genome-wide association studies have linked single-nucleotide polymorphisms (SNPs) in NEMP1 (nuclear envelope membrane protein 1) with early menopause; however, it is unclear whether NEMP1 has any role in fertility. We show that whole-animal loss of NEMP1 homologs in Drosophila, Caenorhabditis elegans, zebrafish, and mice leads to sterility or early loss of fertility. Loss of Nemp leads to nuclear shaping defects, most prominently in the germ line. Biochemical, biophysical, and genetic studies reveal that NEMP proteins support the mechanical stiffness of the germline nuclear envelope via formation of a NEMP-EMERIN complex. These data indicate that the germline nuclear envelope has specialized mechanical properties and that NEMP proteins play essential and conserved roles in fertility.
RESUMEN
Angiogenesis, new blood vessel formation from preexisting vessels, is critical for solid tumor growth. As such, there have been efforts to inhibit angiogenesis as a means to obstruct tumor growth. However, antiangiogenic therapy faces major challenges to the selective targeting of tumor-associated-vessels, as current antiangiogenic targets also disrupt steady-state vessels. Here, we demonstrate that the developmentally critical transcription factor Etv2 is selectively upregulated in both human and mouse tumor-associated endothelial cells (TAECs) and is required for tumor angiogenesis. Two-photon imaging revealed that Etv2-deficient tumor-associated vasculature remained similar to that of steady-state vessels. Etv2-deficient TAECs displayed decreased Flk1 (also known as Vegfr2) expression, FLK1 activation, and proliferation. Endothelial tube formation, proliferation, and sprouting response to VEGF, but not to FGF2, was reduced in Etv2-deficient ECs. ROS activated Etv2 expression in ECs, and ROS blockade inhibited Etv2 expression in TAECs in vivo. Systemic administration of Etv2 siRNA nanoparticles potently inhibited tumor growth and angiogenesis without cardiovascular side effects. These studies highlight a link among vascular oxidative stress, Etv2 expression, and VEGF response that is critical for tumor angiogenesis. Targeting the ETV2 pathway might offer a unique opportunity for more selective antiangiogenic therapies.
Asunto(s)
Células Endoteliales/metabolismo , Neovascularización Patológica/genética , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/farmacocinética , Animales , Femenino , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Terapia Genética/efectos adversos , Terapia Genética/métodos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Nanopartículas/administración & dosificación , Neovascularización Patológica/terapia , Estrés Oxidativo , ARN Interferente Pequeño/genética , Factores de Transcripción/genética , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Recent studies have established that hematopoietic stem cells (HSCs) are quiescent in homeostatic conditions but undergo extensive cell cycle and expansion upon bone marrow (BM) transplantation or hematopoietic injury. The molecular basis for HSC activation and expansion is not completely understood. In this study, we found that key developmentally critical genes controlling hematopoietic stem and progenitor cell (HSPC) generation were up-regulated in HSPCs upon hematopoietic injury. In particular, we found that the ETS transcription factor Ets variant 2 (Etv2; also known as Er71) was up-regulated by reactive oxygen species in HSPCs and was necessary in a cell-autonomous manner for HSPC expansion and regeneration after BM transplantation and hematopoietic injury. We found c-Kit to be downstream of ETV2. As such, lentiviral c-Kit expression rescued Etv2-deficient HSPC proliferation defects in vitro and in short-term BM transplantation in vivo. These findings demonstrate that Etv2 is an important regulator of hematopoietic regeneration.
Asunto(s)
Hematopoyesis , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Regeneración , Factores de Transcripción/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Fluorouracilo/farmacología , Hematopoyesis/efectos de los fármacos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/efectos de los fármacos , Ratones Noqueados , Proteínas Proto-Oncogénicas c-kit/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Regeneración/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
RATIONALE: Wnt signaling regulates key aspects of diabetic vascular disease. OBJECTIVE: We generated SM22-Cre;LRP6(fl/fl);LDLR(-/-) mice to determine contributions of Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6) in the vascular smooth muscle lineage of male low-density lipoprotein receptor-null mice, a background susceptible to diet (high-fat diet)-induced diabetic arteriosclerosis. METHODS AND RESULTS: As compared with LRP6(fl/fl);LDLR(-/-) controls, SM22-Cre;LRP6(fl/fl);LDLR(-/-) (LRP6-VKO) siblings exhibited increased aortic calcification on high-fat diet without changes in fasting glucose, lipids, or body composition. Pulse wave velocity (index of arterial stiffness) was also increased. Vascular calcification paralleled enhanced aortic osteochondrogenic programs and circulating osteopontin (OPN), a matricellular regulator of arteriosclerosis. Survey of ligands and Frizzled (Fzd) receptor profiles in LRP6-VKO revealed upregulation of canonical and noncanonical Wnts alongside Fzd10. Fzd10 stimulated noncanonical signaling and OPN promoter activity via an upstream stimulatory factor (USF)-activated cognate inhibited by LRP6. RNA interference revealed that USF1 but not USF2 supports OPN expression in LRP6-VKO vascular smooth muscle lineage, and immunoprecipitation confirmed increased USF1 association with OPN chromatin. ML141, an antagonist of cdc42/Rac1 noncanonical signaling, inhibited USF1 activation, osteochondrogenic programs, alkaline phosphatase, and vascular smooth muscle lineage calcification. Mass spectrometry identified LRP6 binding to protein arginine methyltransferase (PRMT)-1, and nuclear asymmetrical dimethylarginine modification was increased with LRP6-VKO. RNA interference demonstrated that PRMT1 inhibits OPN and TNAP, whereas PRMT4 supports expression. USF1 complexes containing the histone H3 asymmetrically dimethylated on Arg-17 signature of PRMT4 are increased with LRP6-VKO. Jmjd6, a demethylase downregulated with LRP6 deficiency, inhibits OPN and TNAP expression, USF1: histone H3 asymmetrically dimethylated on Arg-17 complex formation, and transactivation. CONCLUSIONS: LRP6 restrains vascular smooth muscle lineage noncanonical signals that promote osteochondrogenic differentiation, mediated in part via USF1- and arginine methylation-dependent relays.
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Arteriosclerosis/prevención & control , Calcinosis/prevención & control , Diabetes Mellitus Experimental/complicaciones , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/fisiología , Músculo Liso Vascular/fisiopatología , Miocitos del Músculo Liso/metabolismo , Receptores de LDL/deficiencia , Vía de Señalización Wnt , Animales , Arginina/análogos & derivados , Arginina/metabolismo , Arteriosclerosis/etiología , Arteriosclerosis/metabolismo , Calcinosis/etiología , Calcinosis/metabolismo , Diabetes Mellitus Experimental/patología , Grasas de la Dieta/efectos adversos , Receptores Frizzled/fisiología , Regulación de la Expresión Génica/fisiología , Histonas/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/deficiencia , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Masculino , Ratones , Ratones Noqueados , Miocitos del Músculo Liso/patología , Osteopontina/biosíntesis , Osteopontina/genética , Comunicación Paracrina , Mapeo de Interacción de Proteínas , Proteína-Arginina N-Metiltransferasas/metabolismo , Receptores de Superficie Celular , Receptores de LDL/genética , Factores Estimuladores hacia 5'/fisiología , Rigidez Vascular/fisiologíaRESUMEN
When fed high-fat diets, male LDLR(-/-) mice develop obesity, hyperlipidemia, hyperglycemia, and arteriosclerotic calcification. An osteogenic Msx-Wnt regulatory program is concomitantly upregulated in the vasculature. To better understand the mechanisms of diabetic arteriosclerosis, we generated SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) mice, assessing the impact of Msx1+Msx2 gene deletion in vascular myofibroblast and smooth muscle cells. Aortic Msx2 and Msx1 were decreased by 95% and 34% in SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) animals versus Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) controls, respectively. Aortic calcium was reduced by 31%, and pulse wave velocity, an index of stiffness, was decreased in SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) mice vs. controls. Fasting blood glucose and lipids did not differ, yet SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) siblings became more obese. Aortic adventitial myofibroblasts from SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) mice exhibited reduced osteogenic gene expression and mineralizing potential with concomitant reduction in multiple Wnt genes. Sonic hedgehog (Shh) and Sca1, markers of aortic osteogenic progenitors, were also reduced, paralleling a 78% reduction in alkaline phosphatase (TNAP)-positive adventitial myofibroblasts. RNA interference revealed that although Msx1+Msx2 supports TNAP and Wnt7b expression, Msx1 selectively maintains Shh and Msx2 sustains Wnt2, Wnt5a, and Sca1 expression in aortic adventitial myofibroblast cultures. Thus, Msx1 and Msx2 support vascular mineralization by directing the osteogenic programming of aortic progenitors in diabetic arteriosclerosis.
Asunto(s)
Aorta/metabolismo , Arteriosclerosis/genética , Diabetes Mellitus Experimental/genética , Proteínas de Homeodominio/genética , Factor de Transcripción MSX1/genética , Miocitos del Músculo Liso/metabolismo , Miofibroblastos/metabolismo , Calcificación Vascular/genética , Rigidez Vascular/genética , Animales , Antígenos Ly/metabolismo , Arteriosclerosis/metabolismo , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Dieta Alta en Grasa , Eliminación de Gen , Perfilación de la Expresión Génica , Proteínas Hedgehog/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Osteogénesis/genética , Receptores de LDL/genética , Calcificación Vascular/metabolismo , Proteínas Wnt/metabolismo , Proteína Wnt-5a , Proteína wnt2/metabolismoRESUMEN
OBJECTIVE: Endothelial cells (ECs) can undergo an endothelial-mesenchymal transition with tissue fibrosis. Wnt- and Msx2-regulated signals participate in arteriosclerotic fibrosis and calcification. We studied the impact of Wnt7, Msx2, and Dkk1, a Wnt7 antagonist, on endothelial-mesenchymal transition in primary aortic ECs. APPROACH AND RESULTS: Transduction of aortic ECs with vectors expressing Dkk1 suppressed EC differentiation and induced a mineralizing myofibroblast phenotype. Dkk1 suppressed claudin 5, PECAM, cadherin 5 (Cdh5), Tie1, and Tie2. Dkk1 converted the cuboidal cell monolayer into a spindle-shaped multilayer and inhibited EC cord formation. Myofibroblast and osteogenic markers, SM22, type I collagen, Osx, Runx2, and alkaline phosphatase, were upregulated by Dkk1 via activin-like kinase/Smad pathways. Dkk1 increased fibrotic mineralization of aortic ECs cultured under osteogenic conditions--the opposite of mesenchymal cell responses. Msx2 and Wnt7b maintained morphology and upregulated markers of differentiated ECs. Deleting EC Wnt7b with the Cdh5-Cre transgene in Wnt7b(fl/fl);LDLR(-/-) mice upregulated aortic osteogenic genes (Osx, Sox9, Runx2, and Msx2) and nuclear phospho-Smad1/5, and increased collagen and calcium accumulation. CONCLUSIONS: Dkk1 enhances endothelial-mesenchymal transition in aortic ECs, whereas Wnt7b and Msx2 signals preserve EC phenotype. EC responses to Dkk1, Wnt7b, and Msx2 are the opposite of mesenchymal responses, coupling EC phenotypic stability with osteofibrogenic predilection during arteriosclerosis.
Asunto(s)
Aorta/metabolismo , Células Endoteliales/metabolismo , Transición Epitelial-Mesenquimal , Proteínas de Homeodominio/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Miofibroblastos/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Arteriosclerosis/genética , Arteriosclerosis/metabolismo , Arteriosclerosis/patología , Biomarcadores/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Bovinos , Diferenciación Celular , Forma de la Célula , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/patología , Fibrosis , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Ratones , Ratones Noqueados , Miofibroblastos/patología , Neovascularización Fisiológica , Osificación Heterotópica/metabolismo , Fenotipo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Transducción Genética , Transfección , Proteínas Wnt/deficiencia , Proteínas Wnt/genéticaRESUMEN
In LDLR(-/-) mice fed high-fat diabetogenic diets, osteogenic gene-regulatory programs are ectopically activated in vascular myofibroblasts and smooth muscle cells that promote arteriosclerotic calcium deposition. Msx2-Wnt signaling pathways previously identified as important for craniofacial skeletal development are induced in the vasculature by TNF, a prototypic cytokine mediator of the low-grade systemic inflammation of diabesity. To better understand this biology, we studied TNF actions on Msx2 in aortic myofibroblasts. TNF up-regulated Msx2 mRNA 4-fold within 3 h but did not regulate Msx1. Although IL-1ß could also induce Msx2 expression, TNF-related apoptosis inducing ligand, receptor activator of nuclear factor-κB ligand, and IL-6 were inactive. Inhibition of nicotinamide adenine dinucleotide phosphate oxidase (Nox) activity and genetically induced Nox deficiency (p47phox(-/-)) reduced Msx2 induction, indicating contributions of reactive oxygen species (ROS) and redox signaling. Consistent with this, rotenone, an antagonist of mitochondrial complex I, inhibited TNF induction of Msx2 and Nox2, whereas pyruvate, an anapleurotic mitochondrial metabolic substrate, enhanced induction. Moreover, the glutathione peroxidase-mimetic ebselen abrogated this TNF response. Treatment of aortic myofibroblasts with hydrogen peroxide up-regulated Msx2 mRNA, promoter activity, and DNA-protein interactions. In vivo, SM22-TNF transgenic mice exhibit increased aortic Msx2 with no change in Msx1. Dosing SM22-TNF mice with either 20 ng/g Nox1 + 20 ng/g Nox2 antisense oligonucleotides or low-dose rotenone reduced arterial Msx2 expression. Aortic myofibroblasts from TNFR1(-/-) mice expressed levels of Msx2 that were 5% that of wild-type and were not inducible by TNF. Wnt7b and active ß-catenin levels were also reduced. By contrast, TNF-inducible Msx2 expression was not reduced in TNFR2(-/-) cells. Finally, when cultured under mineralizing conditions, TNFR1(-/-) aortic myofibroblasts exhibited reduced calcification compared with wild-type and TNFR2(-/-) cells. Thus, ROS metabolism contributes to TNF induction of Msx2 and procalcific responses in myofibroblasts via TNFR1. Strategies that reduce vascular Nox- or mitochondrially activated ROS signals may prove useful in mitigating arteriosclerotic calcification.