RESUMO
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.
Assuntos
Líquido Cefalorraquidiano , Vértebras Cervicais , Drenagem , Vasos Linfáticos , Animais , Camundongos , Envelhecimento/metabolismo , Líquido Cefalorraquidiano/metabolismo , Vértebras Cervicais/metabolismo , Células Endoteliais/metabolismo , Fluorescência , Genes Reporter , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Vasos Linfáticos/fisiologia , Miócitos de Músculo Liso/metabolismo , Óxido Nítrico/metabolismo , Nariz/fisiologia , Faringe/metabolismo , Receptores Adrenérgicos alfa/metabolismo , Análise de Célula Única , Transdução de SinaisRESUMO
Endothelial cells of mammalian blood vessels have multiple levels of heterogeneity along the vascular tree and among different organs. Further heterogeneity results from blood flow turbulence and variations in shear stress. In the aorta, vascular endothelial protein tyrosine phosphatase (VE-PTP), which dephosphorylates tyrosine kinase receptor Tie2 in the plasma membrane, undergoes downstream polarization and endocytosis in endothelial cells exposed to laminar flow and high shear stress. VE-PTP sequestration promotes Tie2 phosphorylation at tyrosine992 and endothelial barrier tightening. The present study characterized the heterogeneity of VE-PTP polarization, Tie2-pY992 and total Tie2, and claudin-5 in anatomically defined regions of endothelial cells in the mouse descending thoracic aorta, where laminar flow is variable and IgG extravasation is patchy. We discovered that VE-PTP and Tie2-pY992 had mosaic patterns, unlike the uniform distribution of total Tie2. Claudin-5 at tight junctions also had a mosaic pattern, whereas VE-cadherin at adherens junctions bordered all endothelial cells. Importantly, the amounts of Tie2-pY992 and claudin-5 in aortic endothelial cells correlated with downstream polarization of VE-PTP. VE-PTP and Tie2-pY992 also had mosaic patterns in the vena cava, but claudin-5 was nearly absent and extravasated IgG was ubiquitous. Correlation of Tie2-pY992 and claudin-5 with VE-PTP polarization supports their collective interaction in the regulation of endothelial barrier function in the aorta, yet differences between the aorta and vena cava indicate additional flow-related determinants of permeability. Together, the results highlight new levels of endothelial cell functional mosaicism in the aorta and vena cava, where blood flow dynamics are well known to be heterogeneous.
Assuntos
Células Endoteliais , Proteínas Tirosina Fosfatases , Animais , Camundongos , Aorta , Caderinas/metabolismo , Permeabilidade Capilar , Claudina-5/metabolismo , Células Endoteliais/metabolismo , Imunoglobulina G , Mamíferos/metabolismo , Permeabilidade , Proteínas Tirosina Fosfatases/metabolismoRESUMO
BACKGROUND: Mutations in PIEZO1 (Piezo type mechanosensitive ion channel component 1) cause human lymphatic malformations. We have previously uncovered an ORAI1 (ORAI calcium release-activated calcium modulator 1)-mediated mechanotransduction pathway that triggers lymphatic sprouting through Notch downregulation in response to fluid flow. However, the identity of its upstream mechanosensor remains unknown. This study aimed to identify and characterize the molecular sensor that translates the flow-mediated external signal to the Orai1-regulated lymphatic expansion. METHODS: Various mutant mouse models, cellular, biochemical, and molecular biology tools, and a mouse tail lymphedema model were employed to elucidate the role of Piezo1 in flow-induced lymphatic growth and regeneration. RESULTS: Piezo1 was found to be abundantly expressed in lymphatic endothelial cells. Piezo1 knockdown in cultured lymphatic endothelial cells inhibited the laminar flow-induced calcium influx and abrogated the flow-mediated regulation of the Orai1 downstream genes, such as KLF2 (Krüppel-like factor 2), DTX1 (Deltex E3 ubiquitin ligase 1), DTX3L (Deltex E3 ubiquitin ligase 3L,) and NOTCH1 (Notch receptor 1), which are involved in lymphatic sprouting. Conversely, stimulation of Piezo1 activated the Orai1-regulated mechanotransduction in the absence of fluid flow. Piezo1-mediated mechanotransduction was significantly blocked by Orai1 inhibition, establishing the epistatic relationship between Piezo1 and Orai1. Lymphatic-specific conditional Piezo1 knockout largely phenocopied sprouting defects shown in Orai1- or Klf2- knockout lymphatics during embryo development. Postnatal deletion of Piezo1 induced lymphatic regression in adults. Ectopic Dtx3L expression rescued the lymphatic defects caused by Piezo1 knockout, affirming that the Piezo1 promotes lymphatic sprouting through Notch downregulation. Consistently, transgenic Piezo1 expression or pharmacological Piezo1 activation enhanced lymphatic sprouting. Finally, we assessed a potential therapeutic value of Piezo1 activation in lymphatic regeneration and found that a Piezo1 agonist, Yoda1, effectively suppressed postsurgical lymphedema development. CONCLUSIONS: Piezo1 is an upstream mechanosensor for the lymphatic mechanotransduction pathway and regulates lymphatic growth in response to external physical stimuli. Piezo1 activation presents a novel therapeutic opportunity for preventing postsurgical lymphedema. The Piezo1-regulated lymphangiogenesis mechanism offers a molecular basis for Piezo1-associated lymphatic malformation in humans.
Assuntos
Vasos Linfáticos , Linfedema , Animais , Células Endoteliais/metabolismo , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Vasos Linfáticos/metabolismo , Linfedema/metabolismo , Mecanotransdução Celular/fisiologia , Camundongos , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Despite many reports about pulmonary blood vessels in lung fibrosis, the contribution of lymphatics to fibrosis is unknown. We examined the mechanism and consequences of lymphatic remodeling in mice with lung fibrosis after bleomycin injury or telomere dysfunction. Widespread lymphangiogenesis was observed after bleomycin treatment and in fibrotic lungs of prospero homeobox 1-enhanced green fluorescent protein (Prox1-EGFP) transgenic mice with telomere dysfunction. In loss-of-function studies, blocking antibodies revealed that lymphangiogenesis 14 days after bleomycin treatment was dependent on vascular endothelial growth factor (Vegf) receptor 3 signaling, but not on Vegf receptor 2. Vegfc gene and protein expression increased specifically. Extensive extravasated plasma, platelets, and macrophages at sites of lymphatic growth were potential sources of Vegfc. Lymphangiogenesis peaked at 14 to 28 days after bleomycin challenge, was accompanied by doubling of chemokine (C-C motif) ligand 21 in lung lymphatics and tertiary lymphoid organ formation, and then decreased as lung injury resolved by 56 days. In gain-of-function studies, expansion of the lung lymphatic network by transgenic overexpression of Vegfc in club cell secretory protein (CCSP)/VEGF-C mice reduced macrophage accumulation and fibrosis and accelerated recovery after bleomycin treatment. These findings suggest that lymphatics have an overall protective effect in lung injury and fibrosis and fit with a mechanism whereby lung lymphatic network expansion reduces lymph stasis and increases clearance of fluid and cells, including profibrotic macrophages.
Assuntos
Proliferação de Células/fisiologia , Fibrose/patologia , Lesão Pulmonar/patologia , Linfangiogênese/fisiologia , Fator C de Crescimento do Endotélio Vascular/metabolismo , Animais , Fibrose/metabolismo , Vasos Linfáticos/patologia , Macrófagos/metabolismo , Camundongos Transgênicos , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Chylous pleural effusion (chylothorax) frequently accompanies lymphatic vessel malformations and other conditions with lymphatic defects. Although retrograde flow of chyle from the thoracic duct is considered a potential mechanism underlying chylothorax in patients and mouse models, the path chyle takes to reach the thoracic cavity is unclear. Herein, we use a novel transgenic mouse model, where doxycycline-induced overexpression of vascular endothelial growth factor (VEGF)-C was driven by the adipocyte-specific promoter adiponectin (ADN), to determine how chylothorax forms. Surprisingly, 100% of adult ADN-VEGF-C mice developed chylothorax within 7 days. Rapid, consistent appearance of chylothorax enabled us to examine the step-by-step development in otherwise normal adult mice. Dynamic imaging with a fluorescent tracer revealed that lymph in the thoracic duct of these mice could enter the thoracic cavity by retrograde flow into enlarged paravertebral lymphatics and subpleural lymphatic plexuses that had incompetent lymphatic valves. Pleural mesothelium overlying the lymphatic plexuses underwent exfoliation that increased during doxycycline exposure. Together, the findings indicate that chylothorax in ADN-VEGF-C mice results from retrograde flow of chyle from the thoracic duct into lymphatic tributaries with defective valves. Chyle extravasates from these plexuses and enters the thoracic cavity through exfoliated regions of the pleural mesothelium.
Assuntos
Quilotórax/genética , Sistema Linfático/anormalidades , Fator C de Crescimento do Endotélio Vascular/genética , Animais , Quilotórax/patologia , Vasos Linfáticos/anormalidades , Camundongos , Camundongos TransgênicosRESUMO
Polycystic kidney diseases (PKD) are genetic disorders characterized by progressive epithelial cyst growth leading to destruction of normally functioning renal tissue. Current therapies have focused on the cyst epithelium, and little is known about how the blood and lymphatic microvasculature modulates cystogenesis. Hypomorphic Pkd1(nl/nl) mice were examined, showing that cystogenesis was associated with a disorganized pericystic network of vessels expressing platelet/endothelial cell adhesion molecule 1 and vascular endothelial growth factor receptor 3 (VEGFR3). The major ligand for VEGFR3 is VEGFC, and there were lower levels of Vegfc mRNA within the kidneys during the early stages of cystogenesis in 7-day-old Pkd1(nl/nl) mice. Seven-day-old mice were treated with exogenous VEGFC for 2 weeks on the premise that this would remodel both the VEGFR3(+) pericystic vascular network and larger renal lymphatics that may also affect the severity of PKD. Treatment with VEGFC enhanced VEGFR3 phosphorylation in the kidney, normalized the pattern of the pericystic network of vessels, and widened the large lymphatics in Pkd1(nl/nl) mice. These effects were associated with significant reductions in cystic disease, BUN and serum creatinine levels. Furthermore, VEGFC administration reduced M2 macrophage pericystic infiltrate, which has been implicated in the progression of PKD. VEGFC administration also improved cystic disease in Cys1(cpk/cpk) mice, a model of autosomal recessive PKD, leading to a modest but significant increase in lifespan. Overall, this study highlights VEGFC as a potential new treatment for some aspects of PKD, with the possibility for synergy with current epithelially targeted approaches.
Assuntos
Doenças Renais Policísticas/tratamento farmacológico , Fator C de Crescimento do Endotélio Vascular/uso terapêutico , Animais , Camundongos , Doenças Renais Policísticas/etiologia , Fator C de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Remodeling of blood vessels and lymphatics are prominent features of sustained inflammation. Angiopoietin-2 (Ang2)/Tie2 receptor signaling and tumor necrosis factor-α (TNF)/TNF receptor signaling are known to contribute to these changes in airway inflammation after Mycoplasma pulmonis infection in mice. We determined whether Ang2 and TNF are both essential for the remodeling on blood vessels and lymphatics, and thereby influence the actions of one another. Their respective contributions to the initial stage of vascular remodeling and sprouting lymphangiogenesis were examined by comparing the effects of function-blocking antibodies to Ang2 or TNF, given individually or together during the first week after infection. As indices of efficacy, vascular enlargement, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic vessel sprouting were assessed. Inhibition of Ang2 or TNF alone reduced the remodeling of blood vessels and lymphatics, but inhibition of both together completely prevented these changes. Genome-wide analysis of changes in gene expression revealed synergistic actions of the antibody combination over a broad range of genes and signaling pathways involved in inflammatory responses. These findings demonstrate that Ang2 and TNF are essential and synergistic drivers of remodeling of blood vessels and lymphatics during the initial stage of inflammation after infection. Inhibition of Ang2 and TNF together results in widespread suppression of the inflammatory response.
Assuntos
Infecções por Mycoplasma/patologia , Mycoplasma pulmonis/fisiologia , Ribonuclease Pancreático/antagonistas & inibidores , Transdução de Sinais , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Feminino , Inflamação , Linfangiogênese , Sistema Linfático/metabolismo , Sistema Linfático/patologia , Vasos Linfáticos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Mycoplasma/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Pericitos/patologia , Sistema Respiratório/metabolismo , Sistema Respiratório/patologia , Ribonuclease Pancreático/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
RATIONALE: Lymphatic vessels in the respiratory tract normally mature into a functional network during the neonatal period, but under some pathological conditions they can grow as enlarged, dilated sacs that result in the potentially lethal condition of pulmonary lymphangiectasia. OBJECTIVE: We sought to determine whether overexpression of the lymphangiogenic growth factor (vascular endothelial growth factor-C [VEGF-C]) can promote lymphatic growth and maturation in the respiratory tract. Unexpectedly, perinatal overexpression of VEGF-C in the respiratory epithelium led to a condition resembling human pulmonary lymphangiectasia, a life-threatening disorder of the newborn characterized by respiratory distress and the presence of widely dilated lymphatics. METHODS AND RESULTS: Administration of doxycycline to Clara cell secretory protein-reverse tetracycline-controlled transactivator/tetracycline operator-VEGF-C double-transgenic mice during a critical period from embryonic day 15.5 to postnatal day 14 was accompanied by respiratory distress, chylothorax, pulmonary lymphangiectasia, and high mortality. Enlarged sac-like lymphatics were abundant near major airways, pulmonary vessels, and visceral pleura. Side-by-side comparison revealed morphological features similar to pulmonary lymphangiectasia in humans. The condition was milder in mice given doxycycline after age postnatal day 14 and did not develop after postnatal day 35. Mechanistic studies revealed that VEGF recptor (VEGFR)-3 alone drove lymphatic growth in adult mice, but both VEGFR-2 and VEGFR-3 were required for the development of lymphangiectasia in neonates. VEGFR-2/VEGFR-3 heterodimers were more abundant in the dilated lymphatics, consistent with the involvement of both receptors. Despite the dependence of lymphangiectasia on VEGFR-2 and VEGFR-3, the condition was not reversed by blocking both receptors together or by withdrawing VEGF-C. CONCLUSIONS: The findings indicate that VEGF-C overexpression can induce pulmonary lymphangiectasia during a critical period in perinatal development.
Assuntos
Pneumopatias/congênito , Linfangiectasia/congênito , Fator C de Crescimento do Endotélio Vascular/genética , Animais , Feminino , Humanos , Lactente , Pneumopatias/genética , Pneumopatias/metabolismo , Pneumopatias/patologia , Linfangiectasia/genética , Linfangiectasia/metabolismo , Linfangiectasia/patologia , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patologia , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Gravidez , Edema Pulmonar/genética , Edema Pulmonar/metabolismo , Edema Pulmonar/patologia , Transdução de Sinais/fisiologia , Traqueia/metabolismo , Traqueia/patologia , Uteroglobina/genética , Uteroglobina/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Vascular remodeling is a feature of sustained inflammation in which capillaries enlarge and acquire the phenotype of venules specialized for plasma leakage and leukocyte recruitment. We sought to determine whether neutrophils are required for vascular remodeling in the respiratory tract by using Mycoplasma pulmonis infection as a model of sustained inflammation in mice. The time course of vascular remodeling coincided with the influx of neutrophils during the first few days after infection and peaked at day 5. Depletion of neutrophils with antibody RB6-8C5 or 1A8 reduced neutrophil influx and vascular remodeling after infection by about 90%. Similarly, vascular remodeling after infection was suppressed in Cxcr2(-/-) mice, in which neutrophils adhered to the endothelium of venules but did not extravasate into the tissue. Expression of the venular adhesion molecule P-selectin increased in endothelial cells from day 1 to day 3 after infection, as did expression of the Cxcr2-receptor ligands Cxcl1 and Cxcl2. Tumor necrosis factor α (TNFα) expression increased more than sixfold in the trachea of wild-type and Cxcr2(-/-) mice, but intratracheal administration of TNFα did not induce vascular remodeling similar to that seen in infection. We conclude that neutrophil influx is required for remodeling of capillaries into venules in the airways of mice with Mycoplasma infection and that TNFα signaling is necessary but not sufficient for vascular remodeling.
Assuntos
Endotélio Vascular/metabolismo , Infecções por Mycoplasma/metabolismo , Mycoplasma pulmonis , Neutrófilos/metabolismo , Sistema Respiratório/metabolismo , Remodelação Vascular , Animais , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/metabolismo , Endotélio Vascular/patologia , Feminino , Camundongos , Camundongos Knockout , Infecções por Mycoplasma/genética , Infecções por Mycoplasma/patologia , Neutrófilos/patologia , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Sistema Respiratório/patologiaRESUMO
Lymphatics proliferate, become enlarged, or regress in multiple inflammatory lung diseases in humans. Lymphatic growth and remodeling is known to occur in the mouse trachea in sustained inflammation, but whether intrapulmonary lymphatics exhibit similar plasticity is unknown. We examined the time course, distribution, and dependence on vascular endothelial growth factor receptor (VEGFR)-2/VEGFR-3 signaling of lung lymphatics in sustained inflammation. Lymphatics in mouse lungs were examined under baseline conditions and 3 to 28 days after Mycoplasma pulmonis infection, using prospero heomeobox 1-enhanced green fluorescence protein and VEGFR-3 as markers. Sprouting lymphangiogenesis was evident at 7 days. Lymphatic growth was restricted to regions of bronchus-associated lymphoid tissue (BALT), where VEGF-C-producing cells were scattered in T-cell zones. Expansion of lung lymphatics after infection was reduced 68% by blocking VEGFR-2, 83% by blocking VEGFR-3, and 99% by blocking both receptors. Inhibition of VEGFR-2/VEGFR-3 did not prevent the formation of BALT. Treatment of established infection with oxytetracycline caused BALT, but not the lymphatics, to regress. We conclude that robust lymphangiogenesis occurs in mouse lungs after M. pulmonis infection through a mechanism involving signaling of both VEGFR-2 and VEGFR-3. Expansion of the lymphatic network is restricted to regions of BALT, but lymphatics do not regress when BALT regresses after antibiotic treatment. The lung lymphatic network can thus expand in sustained inflammation, but the expansion is not as reversible as the accompanying inflammation.
Assuntos
Brônquios/patologia , Linfangiogênese , Vasos Linfáticos/patologia , Tecido Linfoide/patologia , Pneumonia/patologia , Animais , Anticorpos Bloqueadores/farmacologia , Brônquios/efeitos dos fármacos , Brônquios/microbiologia , Humanos , Linfangiogênese/efeitos dos fármacos , Vasos Linfáticos/efeitos dos fármacos , Vasos Linfáticos/microbiologia , Tecido Linfoide/efeitos dos fármacos , Tecido Linfoide/microbiologia , Camundongos Endogâmicos C57BL , Infecções por Mycoplasma/complicações , Infecções por Mycoplasma/microbiologia , Infecções por Mycoplasma/patologia , Mycoplasma pulmonis/efeitos dos fármacos , Mycoplasma pulmonis/fisiologia , Pneumonia/complicações , Pneumonia/microbiologia , Transdução de Sinais/efeitos dos fármacos , Organismos Livres de Patógenos Específicos , Fatores de Tempo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
These studies used bi-transgenic Clara cell secretory protein (CCSP)/IL-1ß mice that conditionally overexpress IL-1ß in Clara cells to determine whether IL-1ß can promote angiogenesis and lymphangiogenesis in airways. Doxycycline treatment induced rapid, abundant, and reversible IL-1ß production, influx of neutrophils and macrophages, and conspicuous and persistent lymphangiogenesis, but surprisingly no angiogenesis. Gene profiling showed many up-regulated genes, including chemokines (Cxcl1, Ccl7), cytokines (tumor necrosis factor α, IL-1ß, and lymphotoxin-ß), and leukocyte genes (S100A9, Aif1/Iba1). Newly formed lymphatics persisted after IL-1ß overexpression was stopped. Further studies examined how IL1R1 receptor activation by IL-1ß induced lymphangiogenesis. Inactivation of vascular endothelial growth factor (VEGF)-C and VEGF-D by adeno-associated viral vector-mediated soluble VEGFR-3 (VEGF-C/D Trap) completely blocked lymphangiogenesis, showing its dependence on VEGFR-3 ligands. Consistent with this mechanism, VEGF-C immunoreactivity was present in some Aif1/Iba1-immunoreactive macrophages. Because neutrophils contribute to IL-1ß-induced lung remodeling in newborn mice, we examined their potential role in lymphangiogenesis. Triple-transgenic CCSP/IL-1ß/CXCR2(-/-) mice had the usual IL-1ß-mediated lymphangiogenesis but no neutrophil recruitment, suggesting that neutrophils are not essential. IL1R1 immunoreactivity was found on some epithelial basal cells and neuroendocrine cells, suggesting that these cells are targets of IL-1ß, but was not detected on lymphatics, blood vessels, or leukocytes. We conclude that lymphangiogenesis triggered by IL-1ß overexpression in mouse airways is driven by VEGF-C/D from macrophages, but not neutrophils, recruited by chemokines from epithelial cells that express IL1R1.
Assuntos
Interleucina-1beta/metabolismo , Linfangiogênese , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Traqueia/irrigação sanguínea , Traqueia/patologia , Animais , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Epitélio/metabolismo , Epitélio/patologia , Regulação da Expressão Gênica , Humanos , Hipertrofia , Linfangiogênese/genética , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patologia , Camundongos , Camundongos Transgênicos , Neovascularização Patológica/genética , Neutrófilos/metabolismo , Neutrófilos/patologia , Transporte Proteico , Receptores de Interleucina-1/metabolismo , Receptores de Interleucina-8B/metabolismoRESUMO
The dynamic nature of lymphatic vessels is reflected by structural and functional modifications that coincide with changes in their environment. Lymphatics in the respiratory tract undergo rapid changes around birth, during adaptation to air breathing, when lymphatic endothelial cells develop button-like intercellular junctions specialized for efficient fluid uptake and transport. In inflammatory conditions, lymphatic vessels proliferate and undergo remodeling to accommodate greater plasma leakage and immune cell trafficking. However, the newly formed lymphatics are abnormal, and resolution of inflammation is not accompanied by complete reversal of the lymphatic vessel changes back to the baseline. As the understanding of lymphatic plasticity advances, approaches for eliminating the abnormal vessels and improving the functionality of those that remain move closer to reality. This chapter provides an overview of what is known about lymphatic vessel growth, remodeling, and other forms of plasticity that occur during development or inflammation, with an emphasis on the respiratory tract. Also addressed is the limited reversibility of changes in lymphatics during the resolution of inflammation.
Assuntos
Linfangiogênese , Vasos Linfáticos/embriologia , Vasos Linfáticos/fisiologia , Sistema Respiratório/embriologia , Doenças Respiratórias/fisiopatologia , Animais , Humanos , Sistema Respiratório/fisiopatologiaRESUMO
IL-7 is a required factor for T-cell homeostasis. Because of low expression levels and poor reagent availability, the cellular sources of IL-7 have proven challenging to characterize. In this study, we describe a reporter mouse in which enhanced GFP is expressed from the endogenous Il7 locus. We show that IL-7 is produced by lymphatic endothelial cells (LECs) distributed throughout the systemic lymphatic vasculature as well as by fibroblastic reticular cells, and that phosphorylation of STAT5 in lymphocytes is higher in lymphatics than in blood. Furthermore, in nodes depleted of lymphocytes, Il7 transcription is increased in stromal but not in myeloid subsets. These data support recent findings that lymphocyte homeostasis is influenced by access to secondary lymphoid organs and point to LECs as an important in vivo source of IL-7, bathing trafficking immune cells under both resting and lymphopenic conditions.
Assuntos
Células Endoteliais/imunologia , Interleucina-7/biossíntese , Sistema Linfático/citologia , Linfopenia/imunologia , Animais , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/imunologia , Proteínas de Fluorescência Verde/metabolismo , Interleucina-7/imunologia , Interleucina-7/metabolismo , Linfopenia/metabolismo , Camundongos , Camundongos Transgênicos , Fosforilação , Fator de Transcrição STAT5/metabolismoRESUMO
Meningeal lymphatics are conduits for cerebrospinal fluid drainage to lymphatics and lymph nodes in the neck. In this issue of JEM, Boisserand et al. (https://doi.org/10.1084/jem.20221983) provide evidence that expansion of meningeal lymphatics protects against ischemic stroke.
Assuntos
Vasos Linfáticos , Acidente Vascular Cerebral , Humanos , Sistema Linfático , LinfonodosRESUMO
Cathepsin L (Ctsl) is a proposed therapeutic target to control inflammatory responses in a number of disease states. However, Ctsl is thought to support host defense via its involvement in antigen presentation pathways. Hypothesizing that Ctsl helps combat bacterial infection, we investigated its role in Mycoplasma pulmonis-infected mice as a model of acute and chronic infectious airway inflammation. Responses to the airway inoculation of mycoplasma were compared in Ctsl(-/-) and Ctsl(+/+) mice. After infection, Ctsl(-/-) mice demonstrated more body weight loss, greater mortality (22% versus 0%, respectively), and heavier lungs than Ctsl(+/+) mice, but had smaller bronchial lymph nodes. The burden of live mycoplasma in lungs was 247-fold greater in Ctsl(-/-) mice than in Ctsl(+/+) mice after infection for 3 days. Ctsl(-/-) mice exhibited more severe pneumonia and neutrophil-rich, airway-occlusive exudates, which developed more rapidly than in Ctsl(+/+) mice. Compared with the conspicuous remodeling of lymphatics after infection in Ctsl(+/+) mice, little lymphangiogenesis occurred in Ctsl(-/-) mice, but blood vessel remodeling and tissue inflammation were similarly severe. Titers of mycoplasma-reactive IgM, IgA, and IgG in blood in response to live and heat-killed organisms were similar to those in Ctsl(+/+) mice. However, enzyme-linked immunosorbent spot assays revealed profound reductions in the cellular IFN-γ response to mycoplasma antigen. These findings suggest that Ctsl helps contain mycoplasma infection by supporting lymphangiogenesis and cellular immune responses to infection, and our findings predict that the therapeutic inhibition of Ctsl could increase the severity of mycoplasmal infections.
Assuntos
Catepsina L/imunologia , Expressão Gênica/imunologia , Pulmão/enzimologia , Linfangiogênese/imunologia , Vasos Linfáticos/imunologia , Infecções por Mycoplasma/enzimologia , Doença Aguda , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/sangue , Antígenos de Bactérias/imunologia , Carga Bacteriana , Catepsina L/deficiência , Catepsina L/genética , Doença Crônica , Interferon gama/sangue , Interferon gama/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Camundongos , Infecções por Mycoplasma/imunologia , Infecções por Mycoplasma/microbiologia , Infecções por Mycoplasma/mortalidade , Mycoplasma pulmonis/crescimento & desenvolvimento , Índice de Gravidade de Doença , Análise de SobrevidaRESUMO
Recirculation of fluid and cells through lymphatic vessels plays a key role in normal tissue homeostasis, inflammatory diseases, and cancer. Despite recent advances in understanding lymphatic function (Alitalo, K., T. Tammela, and T.V. Petrova. 2005. Nature. 438:946-953), the cellular features responsible for entry of fluid and cells into lymphatics are incompletely understood. We report the presence of novel junctions between endothelial cells of initial lymphatics at likely sites of fluid entry. Overlapping flaps at borders of oak leaf-shaped endothelial cells of initial lymphatics lacked junctions at the tip but were anchored on the sides by discontinuous button-like junctions (buttons) that differed from conventional, continuous, zipper-like junctions (zippers) in collecting lymphatics and blood vessels. However, both buttons and zippers were composed of vascular endothelial cadherin (VE-cadherin) and tight junction-associated proteins, including occludin, claudin-5, zonula occludens-1, junctional adhesion molecule-A, and endothelial cell-selective adhesion molecule. In C57BL/6 mice, VE-cadherin was required for maintenance of junctional integrity, but platelet/endothelial cell adhesion molecule-1 was not. Growing tips of lymphatic sprouts had zippers, not buttons, suggesting that buttons are specialized junctions rather than immature ones. Our findings suggest that fluid enters throughout initial lymphatics via openings between buttons, which open and close without disrupting junctional integrity, but most leukocytes enter the proximal half of initial lymphatics.
Assuntos
Células Endoteliais/citologia , Células Endoteliais/imunologia , Vasos Linfáticos/citologia , Vasos Linfáticos/imunologia , Animais , Caderinas/metabolismo , Movimento Celular , Células Endoteliais/metabolismo , Vasos Linfáticos/metabolismo , Linfócitos/citologia , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismoRESUMO
PURPOSE: To determine whether intravenous ferumoxytol can be used to effectively label mesenchymal stem cells (MSCs) in vivo and can be used for tracking of stem cell transplants. MATERIALS AND METHODS: This study was approved by the institutional animal care and use committee. Sprague-Dawley rats (6-8 weeks old) were injected with ferumoxytol 48 hours prior to extraction of MSCs from bone marrow. Ferumoxytol uptake by these MSCs was evaluated with fluorescence, confocal, and electron microscopy and compared with results of traditional ex vivo-labeling procedures. The in vivo-labeled cells were subsequently transplanted in osteochondral defects of 14 knees of seven athymic rats and were evaluated with magnetic resonance (MR) imaging up to 4 weeks after transplantation. T2 relaxation times of in vivo-labeled MSC transplants and unlabeled control transplants were compared by using t tests. MR data were correlated with histopathologic results. RESULTS: In vivo-labeled MSCs demonstrated significantly higher ferumoxytol uptake compared with ex vivo-labeled cells. With electron microscopy, iron oxide nanoparticles were localized in secondary lysosomes. In vivo-labeled cells demonstrated significant T2 shortening effects in vitro and in vivo when they were compared with unlabeled control cells (T2 in vivo, 15.4 vs 24.4 msec; P < .05) and could be tracked in osteochondral defects for 4 weeks. Histologic examination confirmed the presence of iron in labeled transplants and defect remodeling. CONCLUSION: Intravenous ferumoxytol can be used to effectively label MSCs in vivo and can be used for tracking of stem cell transplants with MR imaging. This method eliminates risks of contamination and biologic alteration of MSCs associated with ex vivo-labeling procedures.
Assuntos
Rastreamento de Células/métodos , Óxido Ferroso-Férrico/administração & dosagem , Imageamento por Ressonância Magnética/métodos , Transplante de Células-Tronco/métodos , Células-Tronco/citologia , Animais , Separação Celular , Células Cultivadas , Meios de Contraste/administração & dosagem , Ratos , Ratos Sprague-Dawley , Coloração e Rotulagem/métodosRESUMO
Endothelial cells of initial lymphatics have discontinuous button-like junctions (buttons), unlike continuous zipper-like junctions (zippers) of collecting lymphatics and blood vessels. Buttons are thought to act as primary valves for fluid and cell entry into lymphatics. To learn when and how buttons form during development and whether they change in disease, we examined the appearance of buttons in mouse embryos and their plasticity in sustained inflammation. We found that endothelial cells of lymph sacs at embryonic day (E)12.5 and tracheal lymphatics at E16.5 were joined by zippers, not buttons. However, zippers in initial lymphatics decreased rapidly just before birth, as buttons appeared. The proportion of buttons increased from only 6% at E17.5 and 12% at E18.5 to 35% at birth, 50% at postnatal day (P)7, 90% at P28, and 100% at P70. In inflammation, zippers replaced buttons in airway lymphatics at 14 and 28 days after Mycoplasma pulmonis infection of the respiratory tract. The change in lymphatic junctions was reversed by dexamethasone but not by inhibition of vascular endothelial growth factor receptor-3 signaling by antibody mF4-31C1. Dexamethasone also promoted button formation during early postnatal development through a direct effect involving glucocorticoid receptor phosphorylation in lymphatic endothelial cells. These findings demonstrate the plasticity of intercellular junctions in lymphatics during development and inflammation and show that button formation can be promoted by glucocorticoid receptor signaling in lymphatic endothelial cells.
Assuntos
Endotélio Linfático/anatomia & histologia , Infecções por Mycoplasma/patologia , Mycoplasma pulmonis , Envelhecimento/patologia , Animais , Animais Recém-Nascidos , Dexametasona/farmacologia , Dexametasona/uso terapêutico , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Endotélio Linfático/efeitos dos fármacos , Endotélio Linfático/embriologia , Endotélio Linfático/crescimento & desenvolvimento , Feminino , Glucocorticoides/farmacologia , Glucocorticoides/uso terapêutico , Junções Intercelulares/fisiologia , Junções Intercelulares/ultraestrutura , Pulmão/anatomia & histologia , Pulmão/embriologia , Pulmão/crescimento & desenvolvimento , Linfangiogênese/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Infecções por Mycoplasma/tratamento farmacológico , Receptores de Glucocorticoides/metabolismo , Junções Íntimas/metabolismo , Traqueia/anatomia & histologia , Traqueia/efeitos dos fármacos , Traqueia/embriologia , Traqueia/crescimento & desenvolvimentoRESUMO
Therapeutic angiogenesis by delivery of vascular growth factors is an attractive strategy for treating debilitating occlusive vascular diseases, yet clinical trials have thus far failed to show efficacy. As a result, limb amputation remains a common outcome for muscle ischemia due to severe atherosclerotic disease, with an overall incidence of 100 per million people in the United States per year. A challenge has been that the angiogenic master regulator vascular endothelial growth factor (VEGF) induces dysfunctional vessels, if expressed outside of a narrow dosage window. We tested the hypothesis that codelivery of platelet-derived growth factor-BB (PDGF-BB), which recruits pericytes, could induce normal angiogenesis in skeletal muscle irrespective of VEGF levels. Coexpression of VEGF and PDGF-BB encoded by separate vectors in different cells or in the same cells only partially corrected aberrant angiogenesis. In marked contrast, coexpression of both factors in every cell at a fixed relative level via a single bicistronic vector led to robust, uniformly normal angiogenesis, even when VEGF expression was high and heterogeneous. Notably, in an ischemic hindlimb model, single-vector expression led to efficient growth of collateral arteries, revascularization, increased blood flow, and reduced tissue damage. Furthermore, these results were confirmed in a clinically applicable gene therapy approach by adenoviral-mediated delivery of the bicistronic vector. We conclude that coordinated expression of VEGF and PDGF-BB via a single vector constitutes a novel strategy for harnessing the potency of VEGF to induce safe and efficacious angiogenesis.
Assuntos
Neovascularização Fisiológica/fisiologia , Proteínas Proto-Oncogênicas c-sis/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/uso terapêutico , Adenoviridae/genética , Animais , Becaplermina , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos , Células HEK293 , Membro Posterior/irrigação sanguínea , Humanos , Masculino , Camundongos , Camundongos SCID , Músculo Esquelético/irrigação sanguínea , Fator de Crescimento Derivado de Plaquetas/uso terapêutico , Proteínas Proto-Oncogênicas c-sis/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/administração & dosagemRESUMO
Vascular endothelial protein tyrosine phosphatase (VE-PTP) influences endothelial barrier function by regulating the activation of tyrosine kinase receptor Tie2. We determined whether this action is linked to the development of atherosclerosis by examining the influence of arterial shear stress on VE-PTP, Tie2 activation, plasma leakage, and atherogenesis. We found that exposure to high average shear stress led to downstream polarization and endocytosis of VE-PTP accompanied by Tie2 activation at cell junctions. In aortic regions with disturbed flow, VE-PTP was not redistributed away from Tie2. Endothelial cells exposed to high shear stress had greater Tie2 activation and less macromolecular permeability than regions with disturbed flow. Deleting endothelial VE-PTP in VE-PTPiECKO mice increased Tie2 activation and reduced plasma leakage in atheroprone regions. ApoE-/- mice bred with VE-PTPiECKO mice had less plasma leakage and fewer atheromas on a high-fat diet. Pharmacologic inhibition of VE-PTP by AKB-9785 had similar anti-atherogenic effects. Together, the findings identify VE-PTP as a novel target for suppression of atherosclerosis.