RESUMEN
Vascular and nervous systems, two major networks in mammalian bodies, show a high degree of anatomical parallelism and functional crosstalk. During development, neurons guide and attract blood vessels, and consequently this parallelism is established. Here, we identified a noncanonical neurovascular interaction in eye development and disease. VEGFR2, a critical endothelial receptor for VEGF, was more abundantly expressed in retinal neurons than in endothelial cells, including endothelial tip cells. Genetic deletion of VEGFR2 in neurons caused misdirected angiogenesis toward neurons, resulting in abnormally increased vascular density around neurons. Further genetic experiments revealed that this misdirected angiogenesis was attributable to an excessive amount of VEGF protein around neurons caused by insufficient engulfment of VEGF by VEGFR2-deficient neurons. Moreover, absence of neuronal VEGFR2 caused misdirected regenerative angiogenesis in ischemic retinopathy. Thus, this study revealed neurovascular crosstalk and unprecedented cellular regulation of VEGF: retinal neurons titrate VEGF to limit neuronal vascularization. PAPERFLICK:
Asunto(s)
Neovascularización Fisiológica , Neuronas/metabolismo , Retina/crecimiento & desarrollo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Endocitosis , Técnicas de Sustitución del Gen , Ratones , Ratones Noqueados , Neurogénesis , Retina/metabolismo , Retina/patología , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Vascular endothelial growth factor (VEGF) blockers are used widely in clinics to target various types of human cancer. Although VEGF blockers exert marked tumor suppressive effects, the therapeutic effects can be limited. Moreover, accumulating evidence shows that VEGF acts not just on endothelial cells but also on various nonendothelial cells, including tumor and immune cells, suggesting a need to revisit the bona fide action of VEGF on endothelial cells using specific genetic mouse models. Herein, tamoxifen-inducible endothelial-specific knockout mice lacking VEGF receptor 2 (Vegfr2), the major signal transducer for VEGF, were used. The initial event resulting from cessation of endothelial Vegfr2 signaling was vascular truncation and fragmentation, rather than maturation of abnormalized vessels. Although deletion of endothelial Vegfr2 suppressed intratumor hemorrhage, it enhanced hypoxia in tumor cells and reduced the number of infiltrating cytotoxic T cells, suggesting a profound reduction in intratumor blood flow. In various tissues, deletion of endothelial Vegfr2 induced regression of healthy capillaries in intestinal villi, substantiating intestinal perforation, which is one of the most common adverse effects of VEGF blockade in humans. Overall, the data suggest that some of the known effects of VEGF blockers on tumor vessels are caused by partial cessation of VEGF signaling, or by actions on nonendothelial cells. The results increase the understanding of the mechanisms underlying anti-angiogenic therapy.
Asunto(s)
Células Endoteliales , Eliminación de Gen , Melanoma Experimental , Proteínas de Neoplasias , Neovascularización Patológica , Receptor 2 de Factores de Crecimiento Endotelial Vascular , Animales , Hipoxia de la Célula/genética , Células Endoteliales/metabolismo , Células Endoteliales/patología , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Transgénicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Vascular endothelial growth factor (VEGF) is a potent mitogen critical for angiogenesis and organogenesis. Deletion or inhibition of VEGF during development not only profoundly suppresses vascular outgrowth, but significantly affects the development and function of various organs. In the brain, VEGF is thought to not only promote vascular growth, but also directly act on neurons as a neurotrophic factor by activating VEGF receptors. In the present study, we demonstrated that deletion of VEGF using hGfap-Cre line, which recombines genes specifically in cortical and hippocampal neurons, severely impaired brain organization and vascularization of these regions. The mutant mice had motor deficits, with lethality around the time of weaning. Multiple reporter lines indicated that VEGF was highly expressed in neurons, but that its cognate receptors, VEGFR1 and 2 were exclusive to endothelial cells in the brain. In accordance, mice lacking neuronal VEGFR1 and VEGFR2 did not exhibit neuronal deformities or lethality. Taken together, our data suggest that neuron-derived VEGF contributes to cortical and hippocampal development likely through angiogenesis independently of direct neurotrophic effects mediated by VEGFR1 and 2.
Asunto(s)
Hipocampo/crecimiento & desarrollo , Neuronas/metabolismo , Lóbulo Parietal/crecimiento & desarrollo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Alelos , Animales , Células Endoteliales/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Neovascularización Fisiológica/genética , Reacción en Cadena de la Polimerasa , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Tissue macrophages, which are ubiquitously present innate immune cells, play versatile roles in development and organogenesis. During development, macrophages prune transient or unnecessary synapses in neuronal development, and prune blood vessels in vascular development, facilitating appropriate tissue remodeling. In the present study, we identified that macrophages contributed to the development of pupillary morphology. Csf1op/op mutant mice, in which ocular macrophages are nearly absent, exhibited abnormal pupillary edges, with abnormal protrusions of excess iris tissue into the pupillary space. Macrophages located near the pupillary edge engulfed pigmented debris, which likely consisted of unnecessary iris protrusions that emerge during smoothening of the pupillary edge. Indeed, pupillary edge macrophages phenotypically possessed some features of M2 macrophages, consistent with robust tissue engulfment and remodeling activities. Interestingly, protruding irises in Csf1op/op mice were only detected in gaps between regressing blood vessels. Taken together, our findings uncovered a new role for ocular macrophages, demonstrating that this cell population is important for iris pruning during development.
Asunto(s)
Macrófagos/metabolismo , Pupila , Animales , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/citología , Ratones , Ratones MutantesRESUMEN
The placental labyrinth is the interface for gas and nutrient exchange between the embryo and the mother; hence its proper development is essential for embryogenesis. However, the molecular mechanism underlying development of the placental labyrinth, particularly in terms of its endothelial organization, is not well understood. Here, we determined that fibronectin leucine-rich transmembrane protein 2 (FLRT2), a repulsive ligand of the UNC5 receptor family for neurons, is unexpectedly expressed in endothelial cells specifically in the placental labyrinth. Mice lacking FLRT2 in endothelial cells exhibited embryonic lethality at mid-gestation, with systemic congestion and hypoxia. Although they lacked apparent deformities in the embryonic vasculature and heart, the placental labyrinths of these embryos exhibited aberrant alignment of endothelial cells, which disturbed the feto-maternal circulation. Interestingly, this vascular deformity was related to endothelial repulsion through binding to the UNC5B receptor. Our results suggest that the proper organization of the placental labyrinth depends on coordinated inter-endothelial repulsion, which prevents uncontrolled layering of the endothelium.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , Organogénesis , Placenta/embriología , Placenta/metabolismo , Transducción de Señal , Animales , Supervivencia Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Células Endoteliales/metabolismo , Femenino , Eliminación de Gen , Hipoxia/patología , Glicoproteínas de Membrana/deficiencia , Ratones Endogámicos C57BL , Neovascularización Fisiológica , Receptores de Netrina , Placenta/irrigación sanguínea , Placenta/citología , Embarazo , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/metabolismoRESUMEN
Hematopoietic stem cells (HSCs) maintain stemness through various mechanisms that protect against stressful conditions. Heat shock proteins (HSPs) preserve cell homeostasis during stress responses through protein quality control, suggesting that HSPs may safeguard HSCs against numerous traumas. Here, we show that mortalin, a mitochondrial HSP, plays an essential role in maintaining HSC properties by regulating oxidative stress. Mortalin is primarily localized in hematopoietic stem and progenitor cell (HSPC) compartments. In this study, the inhibition of mortalin function caused abnormal reactive oxygen species (ROS) elevation in HSCs and reduced HSC numbers. Knockdown (KD) of mortalin in HSPCs impaired their ability to repopulate and form colonies. Moreover, mortalin-KD HSCs could not maintain quiescence and showed severe downregulation of cyclin-dependent kinase inhibitor- and antioxidant-related genes. Conversely, HSCs that overexpressed mortalin maintained a high reconstitution capacity and low ROS levels. Furthermore, DJ-1, one of the genes responsible for Parkinson's disease, directly bound to mortalin and acted as a negative ROS regulator. Using DJ-1-deficient mice, we demonstrated that mortalin and DJ-1 coordinately maintain normal ROS levels and HSC numbers. Collectively, these results indicate that the mortalin/DJ-1 complex guards against mitochondrial oxidative stress and is indispensable for the maintenance of HSCs.
Asunto(s)
Proteínas Portadoras/fisiología , Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/fisiología , Células Madre Hematopoyéticas/citología , Proteínas Oncogénicas/fisiología , Estrés Oxidativo , Animales , Antioxidantes/química , Proteínas Portadoras/genética , Ciclo Celular , Ensayo de Unidades Formadoras de Colonias , Citometría de Flujo , Proteínas HSP70 de Choque Térmico/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Proteínas Oncogénicas/genética , Peroxirredoxinas , Proteína Desglicasa DJ-1 , Piridinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tiazoles/farmacologíaRESUMEN
Bone marrow endothelial cells (BMECs) play a key role in bone formation and haematopoiesis. Although recent studies uncovered the cellular taxonomy of stromal compartments in the bone marrow (BM), the complexity of BMECs is not fully characterized. In the present study, using single-cell RNA sequencing, we defined a spatial heterogeneity of BMECs and identified a capillary subtype, termed type S (secondary ossification) endothelial cells (ECs), exclusively existing in the epiphysis. Type S ECs possessed unique phenotypic characteristics in terms of structure, plasticity and gene expression profiles. Genetic experiments showed that type S ECs atypically contributed to the acquisition of bone strength by secreting type I collagen, the most abundant bone matrix component. Moreover, these cells formed a distinct reservoir for haematopoietic stem cells. These findings provide the landscape for the cellular architecture in the BM vasculature and underscore the importance of epiphyseal ECs during bone and haematopoietic development.
Asunto(s)
Médula Ósea , Células Endoteliales , Médula Ósea/metabolismo , Células Madre Hematopoyéticas/metabolismo , Células de la Médula Ósea , EpífisisRESUMEN
The skeletal system consists of bones and teeth, both of which are hardened via mineralization to support daily physical activity and mastication. The precise mechanism for this process, especially how blood vessels contribute to tissue mineralization, remains incompletely understood. Here, we established an imaging technique to visualize the 3D structure of the tooth vasculature at a single-cell level. Using this technique combined with single-cell RNA sequencing, we identified a unique endothelial subtype specialized to dentinogenesis, a process of tooth mineralization, termed periodontal tip-like endothelial cells. These capillaries exhibit high angiogenic activity and plasticity under the control of odontoblasts; in turn, the capillaries trigger odontoblast maturation. Metabolomic analysis demonstrated that the capillaries perform the phosphate delivery required for dentinogenesis. Taken together, our data identified the fundamental cell-to-cell communications that orchestrate tooth formation, angiogenic-odontogenic coupling, a distinct mechanism compared to the angiogenic-osteogenic coupling in bones. This mechanism contributes to our understanding concerning the functional diversity of organotypic vasculature.
Asunto(s)
Células Endoteliales , Odontogénesis , Animales , Diferenciación Celular , Ratones , Odontoblastos , Odontogénesis/genética , OsteogénesisRESUMEN
Blood vessel abnormalization alters cancer cell metabolism and promotes cancer dissemination and metastasis. However, the biological features of the abnormalized blood vessels that facilitate cancer progression and whether they can be targeted therapeutically have not been fully investigated. Here, we found that an axon guidance molecule, fibronectin leucine-rich transmembrane protein 2 (FLRT2), is expressed preferentially in abnormalized vessels of advanced colorectal cancers in humans and that its expression correlates negatively with long-term survival. Endothelial cell-specific deletion of Flrt2 in mice selectively pruned abnormalized vessels, resulting in a unique metabolic state termed "oxygen-glucose uncoupling," which suppressed tumor metastasis. Moreover, Flrt2 deletion caused an increase in the number of mature vessels, resulting in a significant increase in the antitumor effects of immune checkpoint blockers. Mechanistically, we found that FLRT2 forms noncanonical interendothelial adhesions that safeguard against oxidative stress through homophilic binding. Together, our results demonstrated the existence of tumor-specific interendothelial adhesions that enable abnormalized vessels to facilitate cancer aggressiveness. Targeting this type of adhesion complex could be a safe and effective therapeutic option to suppress cancer progression.
Asunto(s)
Glicoproteínas de Membrana , Neoplasias , Animales , Células Endoteliales/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones , Neovascularización PatológicaRESUMEN
Blood and lymphatic vessels structurally bear a strong resemblance but never share a lumen, thus maintaining their distinct functions. Although lymphatic vessels initially arise from embryonic veins, the molecular mechanism that maintains separation of these two systems has not been elucidated. Here, we show that genetic deficiency of Folliculin, a tumor suppressor, leads to misconnection of blood and lymphatic vessels in mice and humans. Absence of Folliculin results in the appearance of lymphatic-biased venous endothelial cells caused by ectopic expression of Prox1, a master transcription factor for lymphatic specification. Mechanistically, this phenotype is ascribed to nuclear translocation of the basic helix-loop-helix transcription factor Transcription Factor E3 (TFE3), binding to a regulatory element of Prox1, thereby enhancing its venous expression. Overall, these data demonstrate that Folliculin acts as a gatekeeper that maintains separation of blood and lymphatic vessels by limiting the plasticity of committed endothelial cells.
Asunto(s)
Plasticidad de la Célula , Vasos Linfáticos/embriología , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Supresoras de Tumor/deficiencia , Venas/embriología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Núcleo Celular/metabolismo , Embrión de Mamíferos , Células Endoteliales/metabolismo , Endotelio Linfático/citología , Endotelio Linfático/embriología , Endotelio Vascular/citología , Endotelio Vascular/embriología , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Vasos Linfáticos/citología , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas Proto-Oncogénicas/genética , Interferencia de ARN , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Venas/citologíaRESUMEN
TNF-α is a pleiotropic cytokine that has the potential to induce apoptosis under inflammation. How endothelial cells (ECs) are spared from this fate in inflammatory environments where TNF-α is present is not known. Here, we show that TGF-ß-activated kinase 1 (TAK1) ensures EC survival and maintains vascular integrity upon TNF-α stimulation. Endothelial-specific TAK1 knockout mice exhibit intestinal and liver hemorrhage due to EC apoptosis, leading to vascular destruction and rapid death. This EC apoptosis was induced by TNF-α from myeloid cells responding to intestinal microbiota. TNF-α secretion associated with inflammation also induced vascular defects in inflamed organs. Additionally, we determined that TAK1 deletion in tumor ECs resulted in blood vessel and hence tumor regression. Our results illuminate mechanisms ensuring survival of intestinal and liver ECs under physiological conditions and ECs of other organs under inflammatory conditions that could be exploited for anti-angiogenic therapy to treat cancer.
Asunto(s)
Células Endoteliales/patología , Hepatocitos/citología , Inflamación/patología , Quinasas Quinasa Quinasa PAM/metabolismo , Animales , Apoptosis/fisiología , Ratones Transgénicos , Transducción de Señal/fisiologíaRESUMEN
Vascular development involves not only vascular growth, but also regression of transient or unnecessary vessels. Hyaloid vasculature is the temporary circulatory system in fetal eyes, which spontaneously degenerates when the retinal blood vessels start to grow. Failure of the hyaloid vessels to regress leads to disease in humans, persistent hyperplastic primary vitreous, which causes severe intraocular hemorrhage and impairs visual function. However, the mechanism underlying the endogenous program that mediates spontaneous regression of the hyaloid vessels is not well understood. In this study, we identify a robust switch triggering this program directed by neurons in mice. Marked up-regulation of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) occurs in retinal neurons just after birth via distal-multipotent-mesodermal enhancer, a hemangioblast-specific enhancer of VEGFR2. Genetic deletion of neuronal VEGFR2 interrupts this program, resulting in massive hyaloid vessels that persist even during late postnatal days. This abnormality is caused by excessive VEGF proteins in the vitreous cavity as a result of impairment in the neuronal sequestration of VEGF. Collectively, our data indicate that neurons trigger transition from the fetal to the postnatal circulatory systems in the retina.