ABSTRACT
Cerebrospinal fluid (CSF) in the subarachnoid space around the brain has long been known to drain through the lymphatics to cervical lymph nodes1-17, but the connections and regulation have been challenging to identify. Here, using fluorescent CSF tracers in Prox1-GFP lymphatic reporter mice18, we found that the nasopharyngeal lymphatic plexus is a major hub for CSF outflow to deep cervical lymph nodes. This plexus had unusual valves and short lymphangions but no smooth-muscle coverage, whereas downstream deep cervical lymphatics had typical semilunar valves, long lymphangions and smooth muscle coverage that transported CSF to the deep cervical lymph nodes. α-Adrenergic and nitric oxide signalling in the smooth muscle cells regulated CSF drainage through the transport properties of deep cervical lymphatics. During ageing, the nasopharyngeal lymphatic plexus atrophied, but deep cervical lymphatics were not similarly altered, and CSF outflow could still be increased by adrenergic or nitric oxide signalling. Single-cell analysis of gene expression in lymphatic endothelial cells of the nasopharyngeal plexus of aged mice revealed increased type I interferon signalling and other inflammatory cytokines. The importance of evidence for the nasopharyngeal lymphatic plexus functioning as a CSF outflow hub is highlighted by its regression during ageing. Yet, the ageing-resistant pharmacological activation of deep cervical lymphatic transport towards lymph nodes can still increase CSF outflow, offering an approach for augmenting CSF clearance in age-related neurological conditions in which greater efflux would be beneficial.
Subject(s)
Cerebrospinal Fluid , Cervical Vertebrae , Drainage , Lymphatic Vessels , Animals , Mice , Aging/metabolism , Cerebrospinal Fluid/metabolism , Cervical Vertebrae/metabolism , Endothelial Cells/metabolism , Fluorescence , Genes, Reporter , Interferon Type I/immunology , Interferon Type I/metabolism , Lymphatic Vessels/physiology , Myocytes, Smooth Muscle/metabolism , Nitric Oxide/metabolism , Nose/physiology , Pharynx/metabolism , Receptors, Adrenergic, alpha/metabolism , Single-Cell Analysis , Signal TransductionABSTRACT
Autism spectrum disorders (ASD) frequently accompany macrocephaly, which often involves hydrocephalic enlargement of brain ventricles. Katnal2 is a microtubule-regulatory protein strongly linked to ASD, but it remains unclear whether Katnal2 knockout (KO) in mice leads to microtubule- and ASD-related molecular, synaptic, brain, and behavioral phenotypes. We found that Katnal2-KO mice display ASD-like social communication deficits and age-dependent progressive ventricular enlargements. The latter involves increased length and beating frequency of motile cilia on ependymal cells lining ventricles. Katnal2-KO hippocampal neurons surrounded by enlarged lateral ventricles show progressive synaptic deficits that correlate with ASD-like transcriptomic changes involving synaptic gene down-regulation. Importantly, early postnatal Katnal2 re-expression prevents ciliary, ventricular, and behavioral phenotypes in Katnal2-KO adults, suggesting a causal relationship and a potential treatment. Therefore, Katnal2 negatively regulates ependymal ciliary function and its deletion in mice leads to ependymal ciliary hyperfunction and hydrocephalus accompanying ASD-related behavioral, synaptic, and transcriptomic changes.
Subject(s)
Autism Spectrum Disorder , Cilia , Ependyma , Mice, Knockout , Phenotype , Animals , Male , Mice , Autism Spectrum Disorder/genetics , Autism Spectrum Disorder/metabolism , Autism Spectrum Disorder/physiopathology , Behavior, Animal , Cilia/metabolism , Disease Models, Animal , Ependyma/metabolism , Hippocampus/metabolism , Hydrocephalus/genetics , Hydrocephalus/metabolism , Hydrocephalus/pathology , Hydrocephalus/physiopathology , Katanin/metabolism , Katanin/genetics , Mice, Inbred C57BL , Neurons/metabolism , Synapses/metabolism , Transcriptome/geneticsABSTRACT
Recent work has shown that meningeal lymphatic vessels (mLVs), mainly in the dorsal part of the skull, are involved in the clearance of cerebrospinal fluid (CSF), but the precise route of CSF drainage is still unknown. Here we reveal the importance of mLVs in the basal part of the skull for this process by visualizing their distinct anatomical location and characterizing their specialized morphological features, which facilitate the uptake and drainage of CSF. Unlike dorsal mLVs, basal mLVs have lymphatic valves and capillaries located adjacent to the subarachnoid space in mice. We also show that basal mLVs are hotspots for the clearance of CSF macromolecules and that both mLV integrity and CSF drainage are impaired with ageing. Our findings should increase the understanding of how mLVs contribute to the neuropathophysiological processes that are associated with ageing.
Subject(s)
Cerebrospinal Fluid/metabolism , Glymphatic System/anatomy & histology , Glymphatic System/physiology , Lymphatic Vessels/anatomy & histology , Lymphatic Vessels/physiology , Skull Base/anatomy & histology , Aging/pathology , Aging/physiology , Animals , Endothelial Cells/cytology , Endothelial Cells/pathology , Female , Forkhead Transcription Factors/metabolism , Glymphatic System/cytology , Glymphatic System/pathology , Homeodomain Proteins/metabolism , Lymphatic Vessels/cytology , Lymphatic Vessels/pathology , Lymphedema/metabolism , Lymphedema/pathology , Magnetic Resonance Imaging , Male , Mice , Subarachnoid Space/anatomy & histology , Time Factors , Tumor Suppressor Proteins/metabolism , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-3/metabolismABSTRACT
Fabry disease (FD), a lysosomal storage disorder, is caused by defective α-galactosidase (GLA) activity, which results in the accumulation of globotriaosylceramide (Gb3) in endothelial cells and leads to life-threatening complications such as left ventricular hypertrophy (LVH), renal failure, and stroke. Enzyme replacement therapy (ERT) results in Gb3 clearance; however, because of a short half-life in the body and the high immunogenicity of FD patients, ERT has a limited therapeutic effect, particularly in patients with late-onset disease or progressive complications. Because vascular endothelial cells (VECs) derived from FD-induced pluripotent stem cells display increased thrombospondin-1 (TSP1) expression and enhanced SMAD2 signaling, we screened for chemical compounds that could downregulate TSP1 and SMAD2 signaling. Fasudil reduced the levels of p-SMAD2 and TSP1 in FD-VECs and increased the expression of angiogenic factors. Furthermore, fasudil downregulated the endothelial-to-mesenchymal transition (EndMT) and mitochondrial function of FD-VECs. Oral administration of fasudil to FD mice alleviated several FD phenotypes, including LVH, renal fibrosis, anhidrosis, and heat insensitivity. Our findings demonstrate that fasudil is a novel candidate for FD therapy.
Subject(s)
Fabry Disease , Animals , Mice , Fabry Disease/drug therapy , Fabry Disease/genetics , Endothelial Cells/metabolism , alpha-Galactosidase/genetics , Phenotype , Enzyme Replacement TherapyABSTRACT
SIGNIFICANCE STATEMENT: Mesangial cells (MCs) in the kidney are essential to maintaining glomerular integrity, and their impairment leads to major glomerular diseases including diabetic nephropathy (DN). Although high blood glucose elicits abnormal alterations in MCs, the underlying mechanism is poorly understood. We show that YAP/TAZ are increased in MCs of patients with DN and two animal models of DN. High glucose directly induces activation of YAP/TAZ through the canonical Hippo pathway in cultured MCs. Hyperactivation of YAP/TAZ in mouse MCs recapitulates the hallmarks of DN. Activated YAP/TAZ bind and stabilize N-Myc, one of the Myc family. N-Myc stabilization leads to aberrant enhancement of its transcriptional activity and to MC impairments. Our findings shed light on how high blood glucose in diabetes mellitus leads to DN and support a rationale that lowering blood glucose in diabetes mellitus could delay DN pathogenesis. BACKGROUND: Mesangial cells (MCs) in the kidney are central to maintaining glomerular integrity, and their impairment leads to major glomerular diseases, including diabetic nephropathy (DN). Although high blood glucose elicits abnormal alterations in MCs, the underlying molecular mechanism is poorly understood. METHODS: Immunolocalization of YAP/TAZ and pathological features of PDGFRß + MCs were analyzed in the glomeruli of patients with DN, in Zucker diabetic fatty rats, and in Lats1/2i ΔPß mice. RiboTag bulk-RNA sequencing and transcriptomic analysis of gene expression profiles of the isolated MCs from control and Lats1/2iΔPß mice were performed. Immunoprecipitation analysis and protein stability of N-Myc were performed by the standard protocols. RESULTS: YAP and TAZ, the final effectors of the Hippo pathway, are highly increased in MCs of patients with DN and in Zucker diabetic fatty rats. Moreover, high glucose directly induces activation of YAP/TAZ through the canonical Hippo pathway in cultured MCs. Hyperactivation of YAP/TAZ in mouse model MCs recapitulates the hallmarks of DN, including excessive proliferation of MCs and extracellular matrix deposition, endothelial cell impairment, glomerular sclerosis, albuminuria, and reduced glomerular filtration rate. Mechanistically, activated YAP/TAZ bind and stabilize N-Myc protein, one of the Myc family of oncogenes. N-Myc stabilization leads to aberrant enhancement of its transcriptional activity and eventually to MC impairments and DN pathogenesis. CONCLUSIONS: Our findings shed light on how high blood glucose in diabetes mellitus leads to DN and support a rationale that lowering blood glucose in diabetes mellitus could delay DN pathogenesis.
Subject(s)
Diabetes Mellitus , Diabetic Nephropathies , Rats , Mice , Animals , Mesangial Cells/metabolism , Diabetic Nephropathies/metabolism , Blood Glucose/metabolism , Rats, Zucker , Protein Serine-Threonine Kinases/metabolismABSTRACT
RATIONALE: The Hippo pathway governs cellular differentiation, morphogenesis, and homeostasis, but how it regulates these processes in lymphatic vessels is unknown. OBJECTIVE: We aimed to reveal the role of the final effectors of the Hippo pathway, YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif), in lymphatic endothelial cell (LEC) differentiation, morphogenesis, and homeostasis. METHODS AND RESULTS: During mouse embryonic development, LEC-specific depletion of Yap/Taz disturbed both plexus patterning and valve initiation with upregulated Prox1 (prospero homeobox 1). Conversely, LEC-specific YAP/TAZ hyperactivation impaired lymphatic specification and restricted lymphatic sprouting with profoundly downregulated Prox1. Notably, lymphatic YAP/TAZ depletion or hyperactivation aggravated or attenuated pathological lymphangiogenesis in mouse cornea. Mechanistically, VEGF (vascular endothelial growth factor)-C activated canonical Hippo signaling pathway in LECs. Indeed, repression of PROX1 transcription by YAP/TAZ hyperactivation was mediated by recruitment of NuRD (nucleosome remodeling and histone deacetylase) complex and endogenous binding activity of TEAD (TEA domain family members) to the PROX1 promoter. Furthermore, YAP/TAZ hyperactivation enhanced MYC signaling and inhibited CDKN1C, leading to cell cycle dysregulation and aberrant proliferation. CONCLUSIONS: We find that YAP and TAZ play promoting roles in remodeling lymphatic plexus patterning and postnatal lymphatic valve maintenance by negatively regulating Prox1 expression. We further show that YAP and TAZ act as plastic regulators of lymphatic identity and define the Hippo signaling-mediated PROX1 transcriptional programing as a novel dynamic checkpoint underlying LEC plasticity and pathophysiology.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Endothelial Cells/metabolism , Homeodomain Proteins/metabolism , Lymphangiogenesis , Lymphatic Vessels/metabolism , Phosphoproteins/metabolism , Tumor Suppressor Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Cell Cycle Proteins , Cell Differentiation , Cell Plasticity , Cell Proliferation , Cells, Cultured , Endothelial Cells/pathology , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Humans , Lymphatic Vessels/pathology , Mice, Inbred C57BL , Mice, Knockout , Morphogenesis , Phosphoproteins/genetics , Signal Transduction , Trans-Activators , Tumor Suppressor Proteins/genetics , YAP-Signaling ProteinsABSTRACT
A lacteal is a blunt-ended, long, tube-like lymphatic vessel located in the center of each intestinal villus that provides a unique route for drainage of absorbed lipids from the small intestine. However, key regulators for maintaining lacteal integrity are poorly understood. Here, we explore whether and how the gut microbiota regulates lacteal integrity. Germ depletion by antibiotic treatment triggers lacteal regression during adulthood and delays lacteal maturation during the postnatal period. In accordance with compromised lipid absorption, the button-like junction between lymphatic endothelial cells, which is ultrastructurally open to permit free entry of dietary lipids into lacteals, is significantly reduced in lacteals of germ-depleted mice. Lacteal defects are also found in germ-free mice, but conventionalization of germ-free mice leads to normalization of lacteals. Mechanistically, VEGF-C secreted from villus macrophages upon MyD88-dependent recognition of microbes and their products is a main factor in lacteal integrity. Collectively, we conclude that the gut microbiota is a crucial regulator for lacteal integrity by endowing its unique microenvironment and regulating villus macrophages in small intestine.
Subject(s)
Gastrointestinal Microbiome , Intestinal Mucosa/metabolism , Intestinal Mucosa/microbiology , Macrophages/metabolism , Vascular Endothelial Growth Factor C/biosynthesis , Age Factors , Animals , Biological Transport , Biomarkers , CX3C Chemokine Receptor 1/metabolism , Fluorescent Antibody Technique , Intestinal Absorption , Intestinal Mucosa/cytology , Intestinal Mucosa/ultrastructure , Lipid Metabolism , Mice , Microvessels/metabolism , Myeloid Differentiation Factor 88/metabolism , Signal TransductionABSTRACT
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is causing a global crisis. It is still unresolved. Although many therapies and vaccines are being studied, they are still in their infancy. As this pandemic continues, rapid and accurate research for the development of therapies and vaccines is needed. Therefore, it is necessary to understand characteristics of diseases caused by SARS-CoV-2 through animal models. Syrian hamsters are known to be susceptible to SARS-CoV-2. They were intranasally inoculated with SARS-CoV-2. At 2, 4, 8, 12, and 16 days post-infection (dpi), these hamsters were euthanized, and tissues were collected for ultrastructural and microstructural examinations. Microscopic lesions were prominent in the upper and lower respiratory tracts from 2 and 4 dpi groups, respectively. The respiratory epithelium in the trachea, bronchiole, and alveolar showed pathological changes. Inflammatory cells including neutrophils, lymphocytes, macrophages, and eosinophils were infiltrated in/around tracheal lamina propria, pulmonary vessels, alveoli, and bronchiole. In pulmonary lesions, alveolar wall was thickened with infiltrated inflammatory cells, mainly neutrophils and macrophages. In the trachea, epithelial damages started from 2 dpi and recovered from 8 dpi, consistent with microscopic results, High levels of SARS-CoV-2 nucleoprotein were detected at 2 dpi and 4 dpi. In the lung, lesions were most severe at 8 dpi. Meanwhile, high levels of SARS-CoV-2 were detected at 4 dpi. Electron microscopic examinations revealed cellular changes in the trachea epithelium and alveolar epithelium such as vacuolation, sparse micro-organelle, and poor cellular margin. In the trachea epithelium, the number of cytoplasmic organelles was diminished, and small vesicles were prominent from 2 dpi. Some of these electron-lucent vesicles were filled with virion particles. From 8 dpi, the trachea epithelium started to recover. Because of shrunken nucleus and swollen cytoplasm, the N/C ratio of type 2 pneumocyte decreased at 8 and 12 dpi. From 8 dpi, lamellar bodies on type 2 pneumocyte cytoplasm were increasingly observed. Their number then decreased from 16 dpi. However, there was no significant change in type 1 pneumocyte. Viral vesicles were only observed in the cytoplasm of type 2 pneumocyte. In conclusion, ultra- and micro-structural changes presented in this study may provide useful information for SARS-CoV-2 studies in various fields.
Subject(s)
COVID-19/pathology , Respiratory System/pathology , SARS-CoV-2/pathogenicity , Animals , Cricetinae , Immunohistochemistry/veterinary , Male , Mesocricetus , Pilot Projects , RNA, Viral/chemistry , RNA, Viral/isolation & purification , Real-Time Polymerase Chain Reaction/veterinary , Respiratory System/chemistry , Respiratory System/ultrastructure , Respiratory System/virology , Time Factors , Trachea/pathology , Trachea/ultrastructure , Trachea/virology , Weight LossABSTRACT
Angiogenesis, the growth of blood vessels, is a fundamental biological process that controls embryonic development and is also involved in numerous life-threatening human diseases. Much work in the field of angiogenesis research has centred on the vascular endothelial growth factor (VEGF)-VEGF receptor system. The Tie receptors and their angiopoietin (Ang) ligands have been identified as the second vascular tissue-specific receptor Tyr kinase system. Ang-Tie signalling is essential during embryonic vessel assembly and maturation, and functions as a key regulator of adult vascular homeostasis. The structural characteristics and the spatio-temporal regulation of the expression of receptors and ligands provide unique insights into the functions of this vascular signalling system.
Subject(s)
Angiopoietins/metabolism , Blood Vessels/growth & development , Homeostasis , Morphogenesis , Receptors, TIE/metabolism , Animals , Humans , Models, Biological , Signal TransductionABSTRACT
Lymph node lymphatic vessels (LNLVs) serve as a conduit to drain antigens from peripheral tissues to within the lymph nodes. LNLV density is known to be positively regulated by vascular endothelial growth factors secreted by B cells, macrophages, and dendritic cells (DCs). Here, we show that LNLV formation was negatively regulated by T cells. In both steady and inflammatory states, the density of LNLVs was increased in the absence of T cells but decreased when T cells were restored. Interferon-γ secretion by T cells suppressed lymphatic-specific genes in lymphatic endothelial cells and consequently caused marked reduction in LNLV formation. When T cells were depleted, recruitment of antigen-carrying DCs to LNs was augmented, reflecting a compensatory mechanism for antigen presentation to T cells through increased LNLVs. Thus, T cells maintain the homeostatic balance of LNLV density through a negative paracrine action of interferon-γ.
Subject(s)
Dendritic Cells/metabolism , Endothelium, Lymphatic/metabolism , Interferon-gamma/metabolism , Lymphatic Vessels/pathology , T-Lymphocytes/metabolism , Animals , Antigen Presentation/genetics , Cell Movement/genetics , Dendritic Cells/cytology , Dendritic Cells/immunology , Endothelium, Lymphatic/immunology , Endothelium, Lymphatic/pathology , Feedback, Physiological , Interferon-gamma/genetics , Interferon-gamma/immunology , Lymph Nodes/pathology , Lymphangiogenesis/genetics , Lymphatic Vessels/metabolism , Lymphocyte Depletion , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mice, Nude , Paracrine Communication/genetics , Paracrine Communication/immunology , T-Lymphocytes/cytology , T-Lymphocytes/immunologyABSTRACT
The lung is highly vulnerable during sepsis, yet its functional deterioration accompanied by disturbances in the pulmonary microcirculation is poorly understood. This study aimed to investigate how the pulmonary microcirculation is distorted in sepsis-induced acute lung injury (ALI) and reveal the underlying cellular pathophysiologic mechanism.Using a custom-made intravital lung microscopic imaging system in a murine model of sepsis-induced ALI, we achieved direct real-time visualisation of the pulmonary microcirculation and circulating cells in vivo We derived the functional capillary ratio (FCR) as a quantitative parameter for assessing the fraction of functional microvasculature in the pulmonary microcirculation and dead space.We identified that the FCR rapidly decreases in the early stage of sepsis-induced ALI. The intravital imaging revealed that this decrease resulted from the generation of dead space, which was induced by prolonged neutrophil entrapment within the capillaries. We further showed that the neutrophils had an extended sequestration time and an arrest-like dynamic behaviour, both of which triggered neutrophil aggregates inside the capillaries and arterioles. Finally, we found that Mac-1 (CD11b/CD18) was upregulated in the sequestered neutrophils and that a Mac-1 inhibitor restored the FCR and improved hypoxaemia.Using the intravital lung imaging system, we observed that Mac-1-upregulated neutrophil aggregates led to the generation of dead space in the pulmonary microcirculation that was recovered by a Mac-1 inhibitor in sepsis-induced ALI.
Subject(s)
Acute Lung Injury/etiology , Lung/blood supply , Macrophage-1 Antigen/immunology , Neutrophils/cytology , Sepsis/complications , Acute Lung Injury/pathology , Acute Lung Injury/prevention & control , Animals , Antibodies, Monoclonal/pharmacology , Capillaries , Disease Models, Animal , Lung/diagnostic imaging , Male , Mice , Mice, Inbred C57BL , Microcirculation , Microscopy, Video , Sepsis/drug therapy , Sepsis/pathologyABSTRACT
Cellular antioxidant enzymes play crucial roles in aerobic organisms by eliminating detrimental oxidants and maintaining the intracellular redox homeostasis. Therefore, the function of antioxidant enzymes is inextricably linked to the redox-dependent activities of multiple proteins and signaling pathways. Here, we report that the VEGFR2 RTK has an oxidation-sensitive cysteine residue whose reduced state is preserved specifically by peroxiredoxin II (PrxII) in vascular endothelial cells. In the absence of PrxII, the cellular H(2)O(2) level is markedly increased and the VEGFR2 becomes inactive, no longer responding to VEGF stimulation. Such VEGFR2 inactivation is due to the formation of intramolecular disulfide linkage between Cys1199 and Cys1206 in the C-terminal tail. Interestingly, the PrxII-mediated VEGFR2 protection is achieved by association of two proteins in the caveolae. Furthermore, PrxII deficiency suppresses tumor angiogenesis in vivo. This study thus demonstrates a physiological function of PrxII as the residential antioxidant safeguard specific to the redox-sensitive VEGFR2.
Subject(s)
Antioxidants/metabolism , Aorta/enzymology , Endothelial Cells/enzymology , Endothelium, Vascular/enzymology , Neovascularization, Pathologic/enzymology , Peroxiredoxins , Vascular Endothelial Growth Factor Receptor-2 , Animals , Aorta/cytology , Carcinoma, Lewis Lung/enzymology , Carcinoma, Lewis Lung/pathology , Caveolae/enzymology , Cysteine/chemistry , Cysteine/metabolism , Disulfides/chemistry , Disulfides/metabolism , Endothelial Cells/cytology , Endothelium, Vascular/cytology , Gene Silencing , Humans , Hydrogen Peroxide/metabolism , Mice , Mice, Knockout , Mutagenesis, Site-Directed , Neoplasm Transplantation , Neovascularization, Pathologic/genetics , Oxidation-Reduction , Peroxiredoxins/antagonists & inhibitors , Peroxiredoxins/genetics , Peroxiredoxins/metabolism , Protein Binding , Protein Structure, Tertiary , RNA, Small Interfering , Reactive Oxygen Species/metabolism , Signal Transduction , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
Despite the growing interest in adipose tissue as a therapeutic target of metabolic diseases, the identity of adipocyte precursor cells (preadipocytes) and the formation of adipose tissue during embryonic development are still poorly understood. Here, we clarified the identity and dynamic processes of preadipocytes in mouse white adipose tissue during embryogenesis through direct examination, lineage tracing and culture systems. Surprisingly, we found that lipid-lacking but perilipin(+) or adiponectin(+) proliferating preadipocytes started to emerge at embryonic day 16.5, and these cells underwent active proliferation until birth. Moreover, these preadipocytes resided as clusters and were distributed along growing adipose vasculatures. Importantly, the embryonic preadipocytes exhibited considerable coexpression of stem cell markers, such as CD24, CD29 and PDGFRα, and a small portion of preadipocytes were derived from PDGFRß(+) mural cells, in contrast to the adult preadipocytes present in the stromal vascular fraction. Further analyses with in vitro and ex vivo culture systems revealed a stepwise but dynamic regulation of preadipocyte formation and differentiation during prenatal adipogenesis. To conclude, we unraveled the identity and characteristics of embryonic preadipocytes, which are crucial for the formation and expansion of adipose tissue during embryogenesis.
Subject(s)
Adipocytes/metabolism , Adipose Tissue/embryology , Carrier Proteins/metabolism , Cell Proliferation/physiology , Phosphoproteins/metabolism , 3T3-L1 Cells , Adipose Tissue/blood supply , Animals , Azo Compounds , CD24 Antigen/metabolism , Colony-Forming Units Assay , Flow Cytometry , Galactosides , Indoles , Integrin beta1/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Confocal , Perilipin-1 , Real-Time Polymerase Chain Reaction , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Statistics, NonparametricABSTRACT
RATIONALE: Vascular endothelial growth factor (VEGF) signaling is a key pathway for angiogenesis and requires highly coordinated regulation. Although the Notch pathway-mediated suppression of excessive VEGF activity via negative feedback is well known, the positive feedback control for augmenting VEGF signaling remains poorly understood. Transcription factor Sox17 is indispensable for angiogenesis, but its association with VEGF signaling is largely unknown. The contribution of other Sox members to angiogenesis also remains to be determined. OBJECTIVE: To reveal the genetic interaction of Sox7, another Sox member, with Sox17 in developmental angiogenesis and their functional relationship with VEGF signaling. METHODS AND RESULTS: Sox7 is expressed specifically in endothelial cells and its global and endothelial-specific deletion resulted in embryonic lethality with severely impaired angiogenesis in mice, substantially overlapping with Sox17 in both expression and function. Interestingly, compound heterozygosity for Sox7 and Sox17 phenocopied vascular defects of Sox7 or Sox17 homozygous knockout, indicating that the genetic cooperation of Sox7 and Sox17 is sensitive to their combined gene dosage. VEGF signaling upregulated both Sox7 and Sox17 expression in angiogenesis via mTOR pathway. Furthermore, Sox7 and Sox17 promoted VEGFR2 (VEGF receptor 2) expression in angiogenic vessels, suggesting a positive feedback loop between VEGF signaling and SoxF. CONCLUSIONS: Our findings demonstrate that SoxF transcription factors are indispensable players in developmental angiogenesis by acting as positive feedback regulators of VEGF signaling.
Subject(s)
Human Umbilical Vein Endothelial Cells/metabolism , Neovascularization, Physiologic/physiology , SOXF Transcription Factors/physiology , Signal Transduction/physiology , Vascular Endothelial Growth Factor A/metabolism , Animals , Culture Techniques , Female , Humans , Mice , Mice, Knockout , Mice, Transgenic , PregnancyABSTRACT
Penile erection is a neurovascular phenomenon, and erectile dysfunction (ED) is caused mainly by vascular risk factors or diseases, neurologic abnormalities, and hormonal disturbances. Men with diabetic ED often have severe endothelial dysfunction and peripheral nerve damage, which result in poor response to oral phosphodiesterase-5 inhibitors. Nerve injury-induced protein 1 (Ninjurin 1, Ninj1) is known to be involved in neuroinflammatory processes and to be related to vascular regression during the embryonic period. Here, we demonstrate in streptozotocin-induced diabetic mice that inhibition of the Ninj1 pathway by administering Ninj1-neutralizing antibody (Ninj1-Ab) or by using Ninj1-knockout mice successfully restored erectile function through enhanced penile angiogenesis and neural regeneration. Angiopoietin-1 (Ang1) expression was down-regulated and angiopoietin-2 expression was up-regulated in the diabetic penis compared with that in controls, and these changes were reversed by treatment with Ninj1-Ab. Ninj1 blockade-mediated penile angiogenesis and neural regeneration as well as recovery of erectile function were abolished by inhibition of Ang1-Tie2 (tyrosine kinase with Ig and epidermal growth factor homology domain-2) signaling with soluble Tie2 antibody or Ang1 siRNA. The present results suggest that inhibition of the Ninj1 pathway will be a novel therapeutic strategy for treating ED.
Subject(s)
Antibodies, Neutralizing/pharmacology , Cell Adhesion Molecules, Neuronal/antagonists & inhibitors , Diabetes Complications/drug therapy , Erectile Dysfunction/drug therapy , Neovascularization, Physiologic/physiology , Nerve Growth Factors/antagonists & inhibitors , Nerve Regeneration/physiology , Penile Erection/physiology , Analysis of Variance , Angiopoietin-1/metabolism , Animals , Blotting, Western , Cell Adhesion Molecules, Neuronal/genetics , Cell Adhesion Molecules, Neuronal/immunology , DNA Primers/genetics , Gene Expression Regulation/drug effects , Male , Mice , Mice, Inbred NOD , Mice, Knockout , Neovascularization, Physiologic/drug effects , Nerve Growth Factors/genetics , Nerve Growth Factors/immunology , Nerve Regeneration/drug effects , Oligonucleotide Array Sequence Analysis , Penile Erection/drug effects , Receptor, TIE-2/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Signal Transduction/physiologyABSTRACT
BACKGROUND: Lymph node (LN) formation requires multiple but coordinated signaling from intrinsic and extrinsic cellular components during embryogenesis. However, the contribution and role of lymphatic vessels (LVs) in LN formation and maturation are poorly defined. Here, using lymphatic-specific reporters, Prox1-GFP mice and Vegfc+/LacZ mice, we analyzed migration, assembly, and ingrowth of lymphatic endothelial cells (LECs) in LNs during pre- and postnatal development. RESULTS: Prox1+ LECs form string-like connections rather than lymph sac-like structures until E14.5, but the LEC coverage around LN anlagen completes before birth. Compared to wild-type littermates, Vegfc+/LacZ mice had markedly smaller LNs in neonates and adults, presumably due to the decrease in LTi cell clusters and migrating Prox1+ LECs during embryogenesis. In addition, Vegfc-haploinsufficiency or inhibition of VEGFR3 signaling led to an impairment of LN LV ingrowth, resulting in a significant decrease in LN volume. These data indicate that VEGF-C/VEGFR3 signaling plays a substantial role in normal LN formation through proper migration and organization of LECs. CONCLUSIONS: Taken together, our results provide compelling evidence that the contribution of LVs through VEGF-C/VEGFR3 signaling is critical in LN development and maturation. Developmental Dynamics 245:1189-1197, 2016. © 2016 Wiley Periodicals, Inc.
Subject(s)
Lymph Nodes/embryology , Lymph Nodes/metabolism , Lymphangiogenesis/physiology , Animals , Female , Homeodomain Proteins/metabolism , Lymphangiogenesis/genetics , Lymphatic Vessels/embryology , Lymphatic Vessels/metabolism , Mice , Mice, Mutant Strains , Pregnancy , Tumor Suppressor Proteins/metabolism , Vascular Endothelial Growth Factor C/metabolism , Vascular Endothelial Growth Factor Receptor-3/metabolismABSTRACT
BACKGROUND: Intracranial aneurysm (IA) is a common vascular disorder that frequently leads to fatal vascular rupture. Although various acquired risk factors associated with IA have been identified, the hereditary basis of IA remains poorly understood. As a result, genetically modified animals accurately modeling IA and related pathogenesis have been lacking, and subsequent drug development has been delayed. METHODS AND RESULTS: The transcription factor Sox17 is robustly expressed in endothelial cells of normal intracerebral arteries. The combination of Sox17 deficiency and angiotensin II infusion in mice induces vascular abnormalities closely resembling the cardinal features of IA such as luminal dilation, wall thinning, tortuosity, and subarachnoid hemorrhages. This combination impairs junctional assembly, cell-matrix adhesion, regeneration capacity, and paracrine secretion in endothelial cells of intracerebral arteries, highlighting key endothelial dysfunctions that lead to IA pathogenesis. Moreover, human IA samples showed reduced Sox17 expression and impaired endothelial integrity, further strengthening the applicability of this animal model to clinical settings. CONCLUSIONS: Our findings demonstrate that Sox17 deficiency in mouse can induce IA under hypertensive conditions, suggesting Sox17 deficiency as a potential genetic factor for IA formation. The Sox17-deficient mouse model provides a novel platform to develop therapeutics for incurable IA.
Subject(s)
Endothelium, Vascular/pathology , HMGB Proteins/deficiency , Intracranial Aneurysm/genetics , SOXF Transcription Factors/deficiency , SOXF Transcription Factors/physiology , Adult , Aged , Angiotensin II/toxicity , Animals , Aorta/pathology , Cells, Cultured , Cerebral Arteries/chemistry , Cerebral Arteries/pathology , Cyclin-Dependent Kinase Inhibitor Proteins/biosynthesis , Cyclin-Dependent Kinase Inhibitor Proteins/genetics , Dilatation, Pathologic/genetics , Dilatation, Pathologic/pathology , Disease Models, Animal , Endothelium, Vascular/metabolism , Female , HMGB Proteins/genetics , HMGB Proteins/physiology , Humans , Hypertension/complications , Intracranial Aneurysm/etiology , Intracranial Aneurysm/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Myocytes, Smooth Muscle/chemistry , Paracrine Communication , RNA Interference , SOXF Transcription Factors/analysis , SOXF Transcription Factors/genetics , Specific Pathogen-Free Organisms , Subarachnoid Hemorrhage/etiology , Transcription, Genetic , Up-Regulation , Veins/chemistryABSTRACT
PURPOSE: Hepatic malignancies can easily develop resistance to antiangiogenic therapy, but the underlying mechanism remains poorly understood. This study explores whether antiangiogenic therapy influences the tumor vascular network and/or the vessels feeding the hepatic tumor. METHODS: Mice implanted with Lewis lung carcinoma (LLC) cells were subcutaneously injected 3 times (once every other day starting 1 week after LLC implantation) with either an antiangiogenic agent [vascular endothelial growth factor (VEGF)-Trap] or control agent (bovine serum albumin) at a dose of 25 mg/kg before performing angiography. Hepatic arteriography and portography were performed using a vascular cast method with vascular latex. RESULTS: Arteriography of the control-treated LLC-implanted mice showed marked staining of the mass with a prominent feeding artery, suggesting that the tumor is supplied by arterial perfusion. No significant staining was observed on portography. By contrast, 33% (n = 3/9) of the LLC-implanted mice treated with the antiangiogenic agent VEGF-Trap showed intratumoral staining during portography, indicating that these tumors received perfusion via the portal vein. CONCLUSION: Antiangiogenic treatment can induce rearrangement of the hepatic tumor vascular network to establish communication with the portal vein. This implies that hepatic tumors can develop resistance to antiangiogenic therapy by maintaining perfusion through portal venous perfusion.
Subject(s)
Angiogenesis Inhibitors/administration & dosage , Carcinoma, Lewis Lung/drug therapy , Drug Resistance, Neoplasm , Hepatic Artery , Liver Neoplasms, Experimental/drug therapy , Neovascularization, Pathologic , Portal Vein , Receptors, Vascular Endothelial Growth Factor/administration & dosage , Recombinant Fusion Proteins/administration & dosage , Animals , Carcinoma, Lewis Lung/blood supply , Carcinoma, Lewis Lung/diagnostic imaging , Carcinoma, Lewis Lung/pathology , Cell Line, Tumor , Hepatic Artery/diagnostic imaging , Infusions, Intra-Arterial , Infusions, Intravenous , Liver Neoplasms, Experimental/blood supply , Liver Neoplasms, Experimental/diagnostic imaging , Liver Neoplasms, Experimental/pathology , Male , Mice, Inbred C57BL , Portal Vein/diagnostic imaging , Time FactorsABSTRACT
RATIONALE: The Notch pathway stabilizes sprouting angiogenesis by favoring stalk cells over tip cells at the vascular front. Because tip and stalk cells have different properties in morphology and function, their transcriptional regulation remains to be distinguished. Transcription factor Sox17 is specifically expressed in endothelial cells, but its expression and role at the vascular front remain largely unknown. OBJECTIVE: To specify the role of Sox17 and its relationship with the Notch pathway in sprouting angiogenesis. METHODS AND RESULTS: Endothelial-specific Sox17 deletion reduces sprouting angiogenesis in mouse embryonic and postnatal vascular development, whereas Sox17 overexpression increases it. Sox17 promotes endothelial migration by destabilizing endothelial junctions and rearranging cytoskeletal structure and upregulates expression of several genes preferentially expressed in tip cells. Interestingly, Sox17 expression is suppressed in stalk cells in which Notch signaling is relatively high. Notch activation by overexpressing Notch intracellular domain reduces Sox17 expression both in primary endothelial cells and in retinal angiogenesis, whereas Notch inhibition by delta-like ligand 4 (Dll4) blockade increases it. The Notch pathway regulates Sox17 expression mainly at the post-transcriptional level. Furthermore, endothelial Sox17 ablation rescues vascular network from excessive tip cell formation and hyperbranching under Notch inhibition in developmental and tumor angiogenesis. CONCLUSIONS: Our findings demonstrate that the Notch pathway restricts sprouting angiogenesis by reducing the expression of proangiogenic regulator Sox17.