RESUMEN
The bone marrow microenvironment has a key role in regulating haematopoiesis, but its molecular complexity and response to stress are incompletely understood. Here we map the transcriptional landscape of mouse bone marrow vascular, perivascular and osteoblast cell populations at single-cell resolution, both at homeostasis and under conditions of stress-induced haematopoiesis. This analysis revealed previously unappreciated levels of cellular heterogeneity within the bone marrow niche and resolved cellular sources of pro-haematopoietic growth factors, chemokines and membrane-bound ligands. Our studies demonstrate a considerable transcriptional remodelling of niche elements under stress conditions, including an adipocytic skewing of perivascular cells. Among the stress-induced changes, we observed that vascular Notch delta-like ligands (encoded by Dll1 and Dll4) were downregulated. In the absence of vascular Dll4, haematopoietic stem cells prematurely induced a myeloid transcriptional program. These findings refine our understanding of the cellular architecture of the bone marrow niche, reveal a dynamic and heterogeneous molecular landscape that is highly sensitive to stress and illustrate the utility of single-cell transcriptomic data in evaluating the regulation of haematopoiesis by discrete niche populations.
Asunto(s)
Médula Ósea/irrigación sanguínea , Microambiente Celular , Hematopoyesis , Células Madre Hematopoyéticas , Análisis de la Célula Individual , Nicho de Células Madre , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adipocitos/citología , Adipocitos/metabolismo , Animales , Proteínas de Unión al Calcio/metabolismo , Diferenciación Celular , Linaje de la Célula , Endotelio Vascular/citología , Femenino , Regulación de la Expresión Génica , Hematopoyesis/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Masculino , Ratones , Células Mieloides/citología , Células Mieloides/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , RNA-Seq , Receptores Notch/metabolismo , Nicho de Células Madre/genética , Estrés Fisiológico/genética , Transcriptoma/genéticaRESUMEN
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
In addition to their conventional role as a conduit system for gases, nutrients, waste products or cells, blood vessels in the skeletal system play active roles in controlling multiple aspects of bone formation and provide niches for hematopoietic stem cells that reside within the bone marrow. In addition, recent studies have highlighted roles for blood vessels during bone healing. Here, we provide an overview of the architecture of the bone vasculature and discuss how blood vessels form within bone, how their formation is modulated, and how they function during development and fracture repair.
Asunto(s)
Huesos/irrigación sanguínea , Osteogénesis/fisiología , Animales , Condrocitos/citología , Células Endoteliales/citología , Humanos , Neovascularización Fisiológica/fisiología , Osteoblastos/citología , Osteoclastos/citología , Cicatrización de Heridas/fisiologíaRESUMEN
OBJECTIVES: Monocyte/macrophage recruitment and activation at vascular predilection sites plays a central role in the pathogenesis of atherosclerosis. Heterotrimeric G proteins of the G12/13 family have been implicated in the control of migration and inflammatory gene expression, but their function in myeloid cells, especially during atherogenesis, is unknown. APPROACH AND RESULTS: Mice with myeloid-specific deficiency for G12/13 show reduced atherosclerosis with a clear shift to anti-inflammatory gene expression in aortal macrophages. These changes are because of neither altered monocyte/macrophage migration nor reduced activation of inflammatory gene expression; on the contrary, G12/13-deficient macrophages show an increased nuclear factor-κB-dependent gene expression in the resting state. Chronically increased inflammatory gene expression in resident peritoneal macrophages results in myeloid-specific G12/13-deficient mice in an altered peritoneal micromilieu with secondary expansion of peritoneal B1 cells. Titers of B1-derived atheroprotective antibodies are increased, and adoptive transfer of peritoneal cells from mutant mice conveys atheroprotection to wild-type mice. With respect to the mechanism of G12/13-mediated transcriptional control, we identify an autocrine feedback loop that suppresses nuclear factor-κB-dependent gene expression through a signaling cascade involving sphingosine 1-phosphate receptor subtype 2, G12/13, and RhoA. CONCLUSIONS: Together, these data show that selective inhibition of G12/13 signaling in macrophages can augment atheroprotective B-cell populations and ameliorate atherosclerosis.
Asunto(s)
Aorta/metabolismo , Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Subgrupos de Linfocitos B/metabolismo , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Activación de Macrófagos , Macrófagos Peritoneales/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Traslado Adoptivo , Animales , Aorta/inmunología , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Comunicación Autocrina , Subgrupos de Linfocitos B/inmunología , Células Cultivadas , Modelos Animales de Enfermedad , Retroalimentación Fisiológica , Subunidades alfa de la Proteína de Unión al GTP G12-G13/deficiencia , Subunidades alfa de la Proteína de Unión al GTP G12-G13/genética , Regulación de la Expresión Génica , Mediadores de Inflamación/metabolismo , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/trasplante , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/metabolismo , Receptores de LDL/deficiencia , Receptores de LDL/genética , Receptores de Lisoesfingolípidos/deficiencia , Receptores de Lisoesfingolípidos/genética , Transducción de Señal , Receptores de Esfingosina-1-Fosfato , Transcripción Genética , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoARESUMEN
Myelofibrosis and osteosclerosis are fibrotic diseases disrupting bone marrow function that occur in various leukemias but also in response to non-malignant alterations in hematopoietic cells. Here we show that endothelial cell-specific inactivation of the Lats2 gene, encoding Hippo kinase large tumor suppressor kinase 2, or overexpression of the downstream effector YAP1 induce myofibroblast formation and lead to extensive fibrosis and osteosclerosis, which impair bone marrow function and cause extramedullary hematopoiesis in the spleen. Mechanistically, loss of LATS2 induces endothelial-to-mesenchymal transition, resulting in increased expression of extracellular matrix and secreted signaling molecules. Changes in endothelial cells involve increased expression of serum response factor target genes, and, strikingly, major aspects of the LATS2 mutant phenotype are rescued by inactivation of the Srf gene. These findings identify the endothelium as a driver of bone marrow fibrosis, which improves understanding of myelofibrotic and osteosclerotic diseases, for which drug therapies are currently lacking.
RESUMEN
Bone regeneration requires a well-orchestrated cellular and molecular response including robust vascularization and recruitment of mesenchymal and osteogenic cells. In femoral fractures, angiogenesis and osteogenesis are closely coupled during the complex healing process. Here, we show with advanced longitudinal intravital multiphoton microscopy that early vascular sprouting is not directly coupled to osteoprogenitor invasion during calvarial bone regeneration. Early osteoprogenitors emerging from the periosteum give rise to bone-forming osteoblasts at the injured calvarial bone edge. Microvessels growing inside the lesions are not associated with osteoprogenitors. Subsequently, osteogenic cells collectively invade the vascularized and perfused lesion as a multicellular layer, thereby advancing regenerative ossification. Vascular sprouting and remodeling result in dynamic blood flow alterations to accommodate the growing bone. Single cell profiling of injured calvarial bones demonstrates mesenchymal stromal cell heterogeneity comparable to femoral fractures with increase in cell types promoting bone regeneration. Expression of angiogenesis and hypoxia-related genes are slightly elevated reflecting ossification of a vascularized lesion site. Endothelial Notch and VEGF signaling alter vascular growth in calvarial bone repair without affecting the ossification progress. Our findings may have clinical implications for bone regeneration and bioengineering approaches.
Asunto(s)
Regeneración Ósea , Células Madre Mesenquimatosas , Neovascularización Fisiológica , Osteogénesis , Cráneo , Animales , Regeneración Ósea/fisiología , Ratones , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Masculino , Receptores Notch/metabolismo , Receptores Notch/genética , Ratones Endogámicos C57BL , Transducción de Señal , Femenino , AngiogénesisRESUMEN
Developmental osteogenesis, physiological bone remodelling and fracture healing require removal of matrix and cellular debris. Osteoclasts generated by the fusion of circulating monocytes degrade bone, whereas the identity of the cells responsible for cartilage resorption is a long-standing and controversial question. Here we show that matrix degradation and chondrocyte phagocytosis are mediated by fatty acid binding protein 5-expressing cells representing septoclasts, which have a mesenchymal origin and are not derived from haematopoietic cells. The Notch ligand Delta-like 4, provided by endothelial cells, is necessary for septoclast specification and developmental bone growth. Consistent with the termination of growth, septoclasts disappear in adult and ageing bone, but re-emerge in association with growing vessels during fracture healing. We propose that cartilage degradation is mediated by rare, specialized cells distinct from osteoclasts. Our findings have implications for fracture healing, which is frequently impaired in aging humans.
Asunto(s)
Cartílago/metabolismo , Curación de Fractura/fisiología , Células Madre Mesenquimatosas/metabolismo , Osteoclastos/metabolismo , Osteogénesis/fisiología , Animales , Huesos/citología , Huesos/metabolismo , Huesos/ultraestructura , Cartílago/citología , Células Cultivadas , Condrocitos/citología , Condrocitos/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Femenino , Curación de Fractura/genética , Humanos , Masculino , Células Madre Mesenquimatosas/citología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Inmunoelectrónica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Osteoclastos/citología , Osteogénesis/genética , RNA-Seq/métodosRESUMEN
Bone stroma contributes to the regulation of osteogenesis and hematopoiesis but also to fracture healing and disease processes. Mesenchymal stromal cells from bone (BMSCs) represent a heterogenous mixture of different subpopulations with distinct molecular and functional properties. The lineage relationship between BMSC subsets and their regulation by intrinsic and extrinsic factors are not well understood. Here, we show with mouse genetics, ex vivo cell differentiation assays, and transcriptional profiling that BMSCs from metaphysis (mpMSCs) and diaphysis (dpMSCs) are fundamentally distinct. Fate-tracking experiments and single-cell RNA sequencing indicate that bone-forming osteoblast lineage cells and dpMSCs, including leptin receptor-positive (LepR+) reticular cells in bone marrow, emerge from mpMSCs in the postnatal metaphysis. Finally, we show that BMSC fate is controlled by platelet-derived growth factor receptor ß (PDGFRß) signaling and the transcription factor Jun-B. The sum of our findings improves our understanding of BMSC development, lineage relationships, and differentiation.
Asunto(s)
Desarrollo Óseo , Huesos/citología , Linaje de la Célula , Animales , Animales Recién Nacidos , Huesos/ultraestructura , Diferenciación Celular , Células Endoteliales/citología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/ultraestructura , Ratones Endogámicos C57BL , Especificidad de Órganos , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal , Análisis de la Célula Individual , Células del Estroma/citología , Células del Estroma/ultraestructura , Transcripción GenéticaRESUMEN
Blood vessels are integrated into different organ environments with distinct properties and physiology (Augustin and Koh, 2017). A striking example of organ-specific specialization is the bone vasculature where certain molecular signals yield the opposite effect as in other tissues (Glomski et al., 2011; Kusumbe et al., 2014; Ramasamy et al., 2014). Here, we show that the transcriptional coregulators Yap1 and Taz, components of the Hippo pathway, suppress vascular growth in the hypoxic microenvironment of bone, in contrast to their pro-angiogenic role in other organs. Likewise, the kinase Lats2, which limits Yap1/Taz activity, is essential for bone angiogenesis but dispensable in organs with lower levels of hypoxia. With mouse genetics, RNA sequencing, biochemistry, and cell culture experiments, we show that Yap1/Taz constrain hypoxia-inducible factor 1α (HIF1α) target gene expression in vivo and in vitro. We propose that crosstalk between Yap1/Taz and HIF1α controls angiogenesis depending on the level of tissue hypoxia, resulting in organ-specific biological responses.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células Endoteliales/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neovascularización Fisiológica/genética , Transactivadores/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Ciclo Celular/genética , Hipoxia de la Célula/genética , Vía de Señalización Hippo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Osteogénesis/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Transactivadores/genética , Proteínas Señalizadoras YAPRESUMEN
Stem cells (SCs) receive inductive cues from the surrounding microenvironment and cells. Limited molecular evidence has connected tissue-specific mesenchymal stem cells (MSCs) with mesenchymal transit amplifying cells (MTACs). Using mouse incisor as the model, we discover a population of MSCs neibouring to the MTACs and epithelial SCs. With Notch signaling as the key regulator, we disclose molecular proof and lineage tracing evidence showing the distinct MSCs contribute to incisor MTACs and the other mesenchymal cell lineages. MTACs can feedback and regulate the homeostasis and activation of CL-MSCs through Delta-like 1 homolog (Dlk1), which balances MSCs-MTACs number and the lineage differentiation. Dlk1's function on SCs priming and self-renewal depends on its biological forms and its gene expression is under dynamic epigenetic control. Our findings can be validated in clinical samples and applied to accelerate tooth wound healing, providing an intriguing insight of how to direct SCs towards tissue regeneration.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Incisivo/citología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Diferenciación Celular , Linaje de la Célula , Dentina , Epigenómica , Femenino , Expresión Génica , Homeostasis , Humanos , Células Madre Mesenquimatosas/citología , Ratones , Ratones Noqueados , Modelos Animales , Tercer Molar , Ratas , Ratas Wistar , Transducción de Señal , Nicho de Células Madre/fisiología , Cicatrización de HeridasRESUMEN
Measurements of flow velocities at the level of individual arterial vessels and sinusoidal capillaries are crucial for understanding the dynamics of hematopoietic stem and progenitor cell homing in the bone marrow vasculature. We have developed two complementary intravital two-photon imaging approaches to determine blood flow dynamics and velocities in multiple vessel segments by capturing the motion of red blood cells. High-resolution spatiotemporal measurements through a cranial window to determine short-time dynamics of flowing blood cells and repetitive centerline scans were used to obtain a detailed flow-profile map with hemodynamic parameters. In addition, we observed the homing of individual hematopoietic stem and progenitor cells and obtained detailed information on their homing behavior. With our imaging setup, we determined flow patterns at cellular resolution, blood flow velocities and wall shear stress in small arterial vessels and highly branched sinusoidal capillaries, and the cellular dynamics of hematopoietic stem and progenitor cell homing.
Asunto(s)
Velocidad del Flujo Sanguíneo/fisiología , Células de la Médula Ósea/fisiología , Médula Ósea/fisiología , Células Madre Hematopoyéticas/fisiología , Microvasos/fisiología , Animales , Movimiento Celular/fisiología , Hemodinámica/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Resistencia al Corte/fisiología , Estrés Fisiológico/fisiologíaRESUMEN
Blood vessels in the mammalian skeletal system control bone formation and support haematopoiesis by generating local niche environments. While a specialized capillary subtype, termed type H, has been recently shown to couple angiogenesis and osteogenesis in adolescent, adult and ageing mice, little is known about the formation of specific endothelial cell populations during early developmental endochondral bone formation. Here, we report that embryonic and early postnatal long bone contains a specialized endothelial cell subtype, termed type E, which strongly supports osteoblast lineage cells and later gives rise to other endothelial cell subpopulations. The differentiation and functional properties of bone endothelial cells require cell-matrix signalling interactions. Loss of endothelial integrin ß1 leads to endothelial cell differentiation defects and impaired postnatal bone growth, which is, in part, phenocopied by endothelial cell-specific laminin α5 mutants. Our work outlines fundamental principles of vessel formation and endothelial cell differentiation in the developing skeletal system.
Asunto(s)
Huesos/citología , Células Endoteliales/metabolismo , Matriz Extracelular/metabolismo , Osteogénesis , Transducción de Señal , Adipoquinas/metabolismo , Animales , Apelina , Huesos/irrigación sanguínea , Huesos/diagnóstico por imagen , Capilares/citología , Adhesión Celular , Citometría de Flujo , Inmunohistoquímica , Integrasas/metabolismo , Integrina beta1/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones Endogámicos C57BL , Ratones Mutantes , Neovascularización Fisiológica , Fenotipo , Microtomografía por Rayos XRESUMEN
AIMS: VEGF A (VEGF-A) is a central regulator of pre- and postnatal vascular development. In vitro studies suggested that heterotrimeric G-proteins of the Gq/11 family contribute to VEGF receptor 2 (VEGFR2) signalling, but the mechanism and physiological relevance of this finding is unknown. The aim of this study is to understand the role of endothelial Gαq/11 in VEGF-dependent regulation of vascular permeability and angiogenesis. METHODS AND RESULTS: We show here that VEGF-A-induced signalling events, such as VEGFR2 autophosphorylation, calcium mobilization, or phosphorylation of Src and Cdh5, were reduced in Gαq/11-deficient endothelial cells (ECs), resulting in impaired VEGF-dependent barrier opening, tube formation, and proliferation. Agonists at Gq/11-coupled receptors facilitated VEGF-A-induced VEGFR2 autophosphorylation in a Gαq/11-dependent manner, thereby enhancing downstream VEGFR2 signalling. In vivo, EC-specific Gαq/11- and Gαq-deficient mice showed reduced VEGF-induced fluid extravasation, and retinal angiogenesis was significantly impaired. Gαq-deficient ECs showed reduced proliferation, Cdh5 phosphorylation, and fluid extravasation, whereas apoptosis was increased. CONCLUSION: Gαq/11 critically contributes to VEGF-A-dependent permeability control and angiogenic behaviour in vitro and in vivo.
Asunto(s)
Permeabilidad Capilar/efectos de los fármacos , Células Endoteliales/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/fisiología , Neovascularización Fisiológica/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacología , Animales , Permeabilidad Capilar/fisiología , Células Cultivadas , Humanos , Ratones , Neovascularización Fisiológica/fisiología , Fosforilación , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Structural cardiac remodeling, including hypertrophy and fibrosis, plays a crucial role in the pathogenesis of heart failure. In vitro studies suggested a role of the small GTPase RhoA in hypertrophic cardiomyocyte growth, but neither the molecular mechanisms leading to RhoA activation nor their relevance in vivo are known. We use here a mass spectrometric approach to identify Rho guanine nucleotide exchange factors (RhoGEFs) activated during cardiac pressure overload in vivo and show that RhoGEF12 is a central player during cardiac remodeling. We show that RhoGEF12 is required for stretch-induced RhoA activation and hypertrophic gene transcription in vitro and that its activation depends on integrin ß1 and heterotrimeric G proteins of the G12/13 family. In vivo, cardiomyocyte-specific deletion of RhoGEF12 protects mice from overload-induced hypertrophy, fibrosis, and development of heart failure. Importantly, in mice with preexisting hypertrophy, induction of RhoGEF12 deficiency protects from cardiac decompensation, resulting in significantly increased long-term survival. Collectively, RhoGEF12 acts as an integrator of stretch-induced signaling cascades in cardiomyocytes and is an interesting new target for therapeutic intervention in patients with pressure overload-induced heart failure.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Integrinas/metabolismo , Proteínas Musculares/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Transducción de Señal , Animales , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cardiomegalia/patología , Células Cultivadas , Fibrosis , Subunidades alfa de la Proteína de Unión al GTP G12-G13/genética , Factores de Intercambio de Guanina Nucleótido/genética , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Integrinas/genética , Ratones , Ratones Noqueados , Proteínas Musculares/genética , Miocardio/patología , Factores de Intercambio de Guanina Nucleótido RhoRESUMEN
Diagnosis of metastatic breast cancer is associated with a very poor prognosis. New therapeutic targets are urgently needed, but their development is hampered by a lack of understanding of the mechanisms leading to tumor metastasis. Exemplifying this is the fact that the approximately 30% of all breast cancers overexpressing the receptor tyrosine kinase ErbB-2 are characterized by high metastatic potential and poor prognosis, but the signaling events downstream of ErbB-2 that drive cancer cell invasion and metastasis remain incompletely understood. Here we show that overexpression of ErbB-2 in human breast cancer cell lines leads to phosphorylation and activation of the semaphorin receptor Plexin-B1. This was required for ErbB-2-dependent activation of the pro-metastatic small GTPases RhoA and RhoC and promoted invasive behavior of human breast cancer cells. In a mouse model of ErbB-2-overexpressing breast cancer, ablation of the gene encoding Plexin-B1 strongly reduced the occurrence of metastases. Moreover, in human patients with ErbB-2-overexpressing breast cancer, low levels of Plexin-B1 expression correlated with good prognosis. Our data suggest that Plexin-B1 represents a new candidate therapeutic target for treating patients with ErbB-2-positive breast cancer.