RESUMO
The thymus, where T lymphocytes develop and mature, is sensitive to insults such as tissue ischemia or injury. The insults can cause thymic atrophy and compromise T-cell development, potentially impairing adaptive immunity. The objective of this study was to investigate whether myocardial infarction (MI) induces thymic injury to impair T lymphopoiesis and to uncover the underlying mechanisms. When compared with sham controls, MI mice at day 7 post-MI exhibited smaller thymus, lower cellularity, as well as less thymocytes at different developmental stages, indicative of T-lymphopoiesis impairment following MI. Accordingly, the spleen of MI mice has less T cells and recent thymic emigrants (RTEs), implying that the thymus of MI mice releases fewer mature thymocytes than sham controls. Interestingly, the secretory function of splenic T cells was not affected by MI. Further experiments showed that the reduction of thymocytes in MI mice was due to increased thymocyte apoptosis. Removal of adrenal glands by adrenalectomy (ADX) prevented MI-induced thymic injury and dysfunction, whereas corticosterone supplementation in ADX + MI mice reinduced thymic injury and dysfunction, indicating that glucocorticoids mediate thymic damage triggered by MI. Eosinophils play essential roles in thymic regeneration postirradiation, and eosinophil-deficient mice exhibit impaired thymic recovery after sublethal irradiation. Interestingly, the thymus was fully regenerated in both wild-type and eosinophil-deficient mice at day 14 post-MI, suggesting that eosinophils are not critical for thymus regeneration post-MI. In conclusion, our study demonstrates that MI-induced glucocorticoids trigger thymocyte apoptosis and impair T lymphopoiesis, resulting in less mature thymocyte release to the spleen.NEW & NOTEWORTHY The thymus is essential for maintaining whole body T-cell output. Thymic injury can adversely affect T lymphopoiesis and T-cell immune response. This study demonstrates that MI induces thymocyte apoptosis and compromises T lymphopoiesis, resulting in fewer releases of mature thymocytes to the spleen. This process is mediated by glucocorticoids secreted by adrenal glands. Therefore, targeting glucocorticoids represents a novel approach to attenuate post-MI thymic injury.
Assuntos
Adrenalectomia , Apoptose , Linfopoese , Camundongos Endogâmicos C57BL , Infarto do Miocárdio , Timo , Animais , Timo/patologia , Timo/imunologia , Timo/efeitos dos fármacos , Infarto do Miocárdio/patologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/fisiopatologia , Masculino , Timócitos/metabolismo , Timócitos/patologia , Timócitos/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Glucocorticoides/farmacologia , Eosinófilos/metabolismo , Eosinófilos/imunologia , Baço/imunologia , Baço/metabolismo , Baço/patologia , Modelos Animais de Doenças , Camundongos , Corticosterona/sangueRESUMO
BACKGROUND: Severe lung infection can lead to brain dysfunction and neurobehavioral disorders. The mechanisms that regulate the lung-brain axis of inflammatory response to respiratory infection are incompletely understood. This study examined the effects of lung infection causing systemic and neuroinflammation as a potential mechanism contributing to blood-brain barrier (BBB) leakage and behavioral impairment. METHODS: Lung infection in mice was induced by instilling Pseudomonas aeruginosa (PA) intratracheally. We determined bacterial colonization in tissue, microvascular leakage, expression of cytokines and leukocyte infiltration into the brain. RESULTS: Lung infection caused alveolar-capillary barrier injury as indicated by leakage of plasma proteins across pulmonary microvessels and histopathological characteristics of pulmonary edema (alveolar wall thickening, microvessel congestion, and neutrophil infiltration). PA also caused significant BBB dysfunction characterized by leakage of different sized molecules across cerebral microvessels and a decreased expression of cell-cell junctions (VE-cadherin, claudin-5) in the brain. BBB leakage peaked at 24 h and lasted for 7 days post-inoculation. Additionally, mice with lung infection displayed hyperlocomotion and anxiety-like behaviors. To test whether cerebral dysfunction was caused by PA directly or indirectly, we measured bacterial load in multiple organs. While PA loads were detected in the lungs up to 7 days post-inoculation, bacteria were not detected in the brain as evidenced by negative cerebral spinal fluid (CSF) cultures and lack of distribution in different brain regions or isolated cerebral microvessels. However, mice with PA lung infection demonstrated increased mRNA expression in the brain of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α), chemokines (CXCL-1, CXCL-2) and adhesion molecules (VCAM-1 and ICAM-1) along with CD11b + CD45+ cell recruitment, corresponding to their increased blood levels of white cells (polymorphonuclear cells) and cytokines. To confirm the direct effect of cytokines on endothelial permeability, we measured cell-cell adhesive barrier resistance and junction morphology in mouse brain microvascular endothelial cell monolayers, where administration of IL-1ß induced a significant reduction of barrier function coupled with tight junction (TJ) and adherens junction (AJ) diffusion and disorganization. Combined treatment with IL-1ß and TNFα augmented the barrier injury. CONCLUSIONS: Lung bacterial infection is associated with BBB disruption and behavioral changes, which are mediated by systemic cytokine release.
Assuntos
Barreira Hematoencefálica , Pseudomonas aeruginosa , Camundongos , Animais , Barreira Hematoencefálica/metabolismo , Pseudomonas aeruginosa/metabolismo , Doenças Neuroinflamatórias , Citocinas/metabolismo , Pulmão , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Some patients with myocardial infarction (MI) exhibit lymphopenia, a reduction in blood lymphocyte count. Moreover, lymphopenia inversely correlates with patient prognosis. The objective of this study was to elucidate the underlying mechanisms that cause lymphopenia after MI. Multiparameter flow cytometric analysis demonstrated that MI induced profound B and T lymphopenia in a mouse model, peaking at day 1 post-MI. The finding that non-MI control and MI mice exhibited similar apoptotic rate for blood B and T lymphocytes argues against apoptosis being essential for MI-induced lymphopenia. Interestingly, the bone marrow in day 1 post-MI mice contained more B and T cells but showed less B- and T-cell proliferation compared with day 0 controls. This suggests that blood lymphocytes may travel to the bone marrow after MI. This was confirmed by adoptive transfer experiments demonstrating that MI caused the loss of transferred lymphocytes in the blood, but the accumulation of transferred lymphocytes in the bone marrow. To elucidate the underlying signaling pathways, ß2-adrenergic receptor or sphingosine-1-phosphate receptor type 1 (S1PR1) was pharmacologically blocked, respectively. ß2-receptor inhibition had no significant effect on blood lymphocyte count, whereas S1PR1 blockade aggravated lymphopenia in MI mice. Furthermore, we discovered that MI-induced glucocorticoid release triggered lymphopenia. This was supported by the findings that adrenalectomy (ADX) completely prevented mice from MI-induced lymphopenia, and supplementation with corticosterone in adrenalectomized MI mice reinduced lymphopenia. In conclusion, our study demonstrates that MI-associated lymphopenia involves lymphocyte redistribution from peripheral blood to the bone marrow, which is mediated by glucocorticoids.NEW & NOTEWORTHY Lymphopenia, a reduction in blood lymphocyte count, is known to inversely correlate with the prognosis for patients with myocardial infarction (MI). However, the underlying mechanisms by which cardiac ischemia induces lymphopenia remain elusive. This study provides the first evidence that MI activates the hypothalamic-pituitary-adrenal (HPA) axis to increase glucocorticoid secretion, and elevated circulating glucocorticoids induce blood lymphocytes trafficking to the bone marrow, leading to lymphopenia.
Assuntos
Linfopenia , Infarto do Miocárdio , Animais , Medula Óssea , Humanos , Contagem de Linfócitos , Linfócitos , Linfopenia/induzido quimicamente , Camundongos , Infarto do Miocárdio/complicaçõesRESUMO
Emerging evidence shows that after injury or infection, the mesenteric lymph acts as a conduit for gut-derived toxic factors to enter the blood circulation, causing systemic inflammation and acute lung injury. Neither the cellular and molecular identity of lymph factors nor their mechanisms of action have been well understood and thus have become a timely topic of investigation. This review will first provide a summary of background knowledge on gut barrier and mesenteric lymphatics, followed by a discussion focusing on the current understanding of potential injurious factors in the lymph and their mechanistic contributions to lung injury. We also examine lymph factors with antiinflammatory properties as well as the bidirectional nature of the gut-lung axis in inflammation.
Assuntos
Trato Gastrointestinal/patologia , Pulmão/patologia , Vasos Linfáticos/patologia , Síndrome de Resposta Inflamatória Sistêmica/patologia , Lesão Pulmonar Aguda/patologia , Animais , HumanosRESUMO
INTRODUCTION: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 virus, is a predominantly respiratory tract infection with the capacity to affect multiple organ systems. Abnormal liver tests, mainly transaminase elevations, have been reported in hospitalized patients. We describe a syndrome of cholangiopathy in patients recovering from severe COVID-19 characterized by marked elevation in serum alkaline phosphatase (ALP) accompanied by evidence of bile duct injury on imaging. METHODS: We conducted a retrospective study of COVID-19 patients admitted to our institution from March 1, 2020, to August 15, 2020, on whom the hepatology service was consulted for abnormal liver tests. Bile duct injury was identified by abnormal liver tests with serum ALP > 3x upper limit of normal and abnormal findings on magnetic resonance cholangiopacreatography. Clinical, laboratory, radiological, and histological findings were recorded in a Research Electronic Data Capture database. RESULTS: Twelve patients were identified, 11 men and 1 woman, with a mean age of 58 years. Mean time from COVID-19 diagnosis to diagnosis of cholangiopathy was 118 days. Peak median serum alanine aminotransferase was 661 U/L and peak median serum ALP was 1855 U/L. Marked elevations of erythrocyte sedimentation rate, C-reactive protein, and D-dimers were common. Magnetic resonance cholangiopacreatography findings included beading of intrahepatic ducts (11/12, 92%), bile duct wall thickening with enhancement (7/12, 58%), and peribiliary diffusion high signal (10/12, 83%). Liver biopsy in 4 patients showed acute and/or chronic large duct obstruction without clear bile duct loss. Progressive biliary tract damage has been demonstrated radiographically. Five patients were referred for consideration of liver transplantation after experiencing persistent jaundice, hepatic insufficiency, and/or recurrent bacterial cholangitis. One patient underwent successful living donor liver transplantation. DISCUSSION: Cholangiopathy is a late complication of severe COVID-19 with the potential for progressive biliary injury and liver failure. Further studies are required to understand pathogenesis, natural history, and therapeutic interventions.
Assuntos
COVID-19/complicações , Colangite Esclerosante/epidemiologia , Doença Hepática Terminal/epidemiologia , Icterícia/epidemiologia , Adulto , Idoso , Alanina Transaminase/sangue , Fosfatase Alcalina/sangue , Ductos Biliares/diagnóstico por imagem , Ductos Biliares/imunologia , Ductos Biliares/patologia , Biópsia , COVID-19/diagnóstico , COVID-19/imunologia , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19 , Colangiopancreatografia por Ressonância Magnética , Colangite Esclerosante/diagnóstico , Colangite Esclerosante/imunologia , Colangite Esclerosante/terapia , Progressão da Doença , Doença Hepática Terminal/diagnóstico , Doença Hepática Terminal/imunologia , Doença Hepática Terminal/cirurgia , Feminino , Humanos , Icterícia/diagnóstico , Icterícia/imunologia , Icterícia/terapia , Testes de Função Hepática , Transplante de Fígado , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Índice de Gravidade de DoençaRESUMO
Extracellular vesicles (EVs) have attracted rising interests in the cardiovascular field not only because they serve as serological markers for circulatory disorders but also because they participate in important physiological responses to stress and inflammation. In the circulation, these membranous vesicles are mainly derived from blood or vascular cells, and they carry cargos with distinct molecular signatures reflecting the origin and activation state of parent cells that produce them, thus providing a powerful tool for diagnosis and prognosis of pathological conditions. Functionally, circulating EVs mediate tissue-tissue communication by transporting bioactive cargos to local and distant sites, where they directly interact with target cells to alter their function. Recent evidence points to the critical contributions of EVs to the pathogenesis of vascular endothelial barrier dysfunction during inflammatory response to injury or infection. In this review, we provide a brief summary of the current knowledge on EV biology and advanced techniques in EV isolation and characterization. This is followed by a discussion focusing on the role and mechanisms of EVs in regulating blood-endothelium interactions and vascular permeability during inflammation. We conclude with a translational perspective on the diagnostic and therapeutic potential of EVs in vascular injury or infectious diseases, such as COVID-19.
Assuntos
Permeabilidade Capilar , Endotélio Vascular/metabolismo , Vesículas Extracelulares/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/metabolismo , Animais , Betacoronavirus/patogenicidade , COVID-19 , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Endotélio Vascular/patologia , Endotélio Vascular/virologia , Vesículas Extracelulares/patologia , Vesículas Extracelulares/virologia , Interações Hospedeiro-Patógeno , Humanos , Inflamação/patologia , Pandemias , Pneumonia Viral/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , SARS-CoV-2 , Transdução de SinaisRESUMO
BACKGROUND: Increased extracellular histones in the bloodstream are known as a biomarker for vascular dysfunction associated with severe trauma or sepsis. There is limited information regarding the pathogenic role of circulating histones in neuroinflammation and cerebrovascular endothelial injury. Particularly, it remains unclear whether histones affect the blood-brain barrier (BBB) permeability function. METHODS: The direct effects of unfractionated histones on endothelial barrier properties were first assessed in brain microvascular endothelial cell monolayers by measuring transendothelial electrical resistance and solute flux. This was followed by in vivo mouse experiments, where BBB function was assessed by quantifying brain tissue accumulation of intravenously injected tracers of different molecular sizes, and comparison was made in mice receiving a sublethal dose of histones versus sterile saline. In parallel, the endothelial barrier ultrastructure was examined in histone- and saline-injected animals under transmission electron microscopy, corresponding to the expression of tight junction and adherens junction proteins. RESULTS: Histones increased paracellular permeability to sodium fluorescein and reduced barrier resistance at 100 µg/mL; these responses were accompanied by discontinuous staining of the tight junction proteins claudin-5 and zona ocludens-1. Interestingly, the effects of histones did not seem to result from cytotoxicity, as evidenced by negative propidium iodide staining. In vivo, histones increased the paracellular permeability of the BBB to small tracers of < 1-kDa, whereas tracers larger than 3-kDa remained impermeable across brain microvessels. Further analysis of different brain regions showed that histone-induced tracer leakage and loss of tight junction protein expression mainly occurred in the hippocampus, but not in the cerebral cortex. Consistently, opening of tight junctions was found in hippocampal capillaries from histone-injected animals. Protein expression levels of GFAP and iBA1 remained unchanged in histone-injected mice indicating that histones did not affect reactive gliosis. Moreover, cell membrane surface charge alterations are involved in histone-induced barrier dysfunction and tight junction disruption. CONCLUSIONS: Extracellular histones cause a reversible, region-specific increase in BBB permeability to small molecules by disrupting tight junctions in the hippocampus. We suggest that circulating histones may contribute to cerebrovascular injury or brain dysfunction by altering BBB structure and function.
Assuntos
Barreira Hematoencefálica/metabolismo , Permeabilidade Capilar/fisiologia , Líquido Extracelular/metabolismo , Histonas/sangue , Microvasos/metabolismo , Animais , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/efeitos dos fármacos , Permeabilidade Capilar/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Líquido Extracelular/citologia , Líquido Extracelular/efeitos dos fármacos , Feminino , Histonas/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/citologia , Microvasos/efeitos dos fármacosRESUMO
OBJECTIVE: S1P has known endothelial barrier-protective properties, but whether this extends to the BBB is unclear. We hypothesized that alcohol-induced disruption of brain microvascular endothelial barrier function and junctional protein organization can be ameliorated by S1P treatment. METHODS: Cultured primary HBMEC monolayers were used to characterize endothelial-specific mechanisms of BBB regulation. TER and apparent permeability coefficients for albumin, dextran-4 kDa, and sodium fluorescein were used as indices of barrier function. Junctional localization of Claudin-5, VE-cadherin, and ß-catenin was determined by immunofluorescence confocal microscopy. S1P was applied following treatment with alcohol. RESULTS: Alcohol significantly impaired HBMEC TER. Application of S1P after alcohol treatment resulted in a hastened recovery to the baseline HBMEC TER. Alcohol-treated HBMEC had a significantly higher mean permeability than control that was reversed by S1P. Alcohol caused the formation of gaps between cells. Treatment with S1P (after alcohol) increased junctional localization of VE-Cadherin, Claudin-5, and ß-catenin. CONCLUSIONS: Alcohol impairs the barrier function and junctional organization of HBMEC monolayers. S1P enhanced barrier function and restored junctions in the presence of alcohol, and thus may be useful for restoring BBB function during alcohol intoxication.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/irrigação sanguínea , Endotélio Vascular/química , Etanol/toxicidade , Lisofosfolipídeos/fisiologia , Proteínas Musculares/efeitos dos fármacos , Esfingosina/análogos & derivados , Antígenos CD , Barreira Hematoencefálica/patologia , Caderinas , Células Cultivadas , Claudina-5 , Endotélio Vascular/citologia , Humanos , Microcirculação , Proteínas Musculares/química , Permeabilidade/efeitos dos fármacos , Esfingosina/fisiologia , beta CateninaRESUMO
Circulating components of neutrophil extracellular traps (NETs), especially histones, are associated with tissue injury during inflammatory conditions like sepsis. Commonly used as a NET biomarker, citrullinated histone 3 (H3Cit) may also functionally contribute to the NET-associated inflammatory response. To this end, we sought to examine the role of H3Cit in mediating microvascular endothelial barrier dysfunction. Here we show that H3Cit can directly contribute to inflammatory injury by disrupting the microvascular endothelial barrier. We found that endothelial responses to H3Cit are characterized by cell-cell adherens junction opening and cytoskeleton reorganization with increased F-actin stress fibers. Several signaling pathways often implicated in the transduction of hyperpermeability, such as Rho and MLCK, did not appear to play a major role; however, the adenylyl cyclase activator forskolin blocked the endothelial barrier effect of H3Cit. Taken together, the data suggest that H3Cit-induced endothelial barrier dysfunction may hold promise to treat inflammatory injury.
Assuntos
Histonas/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Animais , Células Cultivadas , Colforsina/farmacologia , Histonas/sangue , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Endothelial cells of the microcirculation form a semi-permeable diffusion barrier between the blood and tissues. This permeability of the endothelium, particularly in the capillaries and postcapillary venules, is a normal physiological function needed for blood-tissue exchange in the microcirculation. During inflammation, microvascular permeability increases dramatically and can lead to tissue edema, which in turn can lead to dysfunction of tissues and organs. The molecular mechanisms that control the barrier function of endothelial cells have been under investigation for several decades and remain an important topic due to the potential for discovery of novel therapeutic strategies to reduce edema. This review highlights current knowledge of the cellular and molecular mechanisms that lead to endothelial hyperpermeability during inflammatory conditions associated with injury and disease. This includes a discussion of recent findings demonstrating temporal protrusions by endothelial cells that may contribute to intercellular junction integrity between endothelial cells and affect the diffusion distance for solutes via the paracellular pathway.
Assuntos
Permeabilidade Capilar , Endotélio Vascular/metabolismo , Animais , Humanos , Microcirculação , Microfluídica/métodos , Microvasos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Choque Hemorrágico/metabolismo , Choque Hemorrágico/patologiaRESUMO
Neutrophils play an essential role in host defense against infection or injury. While neutrophil activation is necessary for pathogen clearance and tissue repair, a hyperactive response can lead to tissue damage and microcirculatory disorders, a process involving complex neutrophil-endothelium cross talk. This review highlights recent research findings about neutrophil-mediated signaling and structural changes, including those induced by neutrophil extracellular traps, which ultimately lead to vascular barrier injury.
Assuntos
Endotélio Vascular/lesões , Armadilhas Extracelulares/fisiologia , Ativação de Neutrófilo , Neutrófilos/fisiologia , Animais , Humanos , Microcirculação , Receptor Cross-Talk , Transdução de SinaisRESUMO
The α-synuclein protein exists in vivo in a variety of covalently modified and aggregated forms associated with Parkinson's disease (PD) pathology. However, the specific proteoform structures involved with neuropathological disease mechanisms are not clearly defined. Since α-synuclein plays a role in presynaptic neurotransmitter release, an in vitro enzyme-based assay was developed to measure glutamate release from mouse forebrain synaptoneurosomes (SNs) enriched in synaptic endings. Glutamate measurements utilizing SNs from various mouse genotypes (WT, over-expressers, knock-outs) suggested a concentration dependence of α-synuclein on calcium/depolarization-dependent presynaptic glutamate release from forebrain terminals. In vitro reconstitution experiments with recombinant human α-synuclein proteoforms including monomers and aggregated forms (fibrils, oligomers) produced further evidence of this functional impact. Notably, brief exogenous applications of fibrillated forms of α-synuclein enhanced SN glutamate release but monomeric forms did not, suggesting preferential membrane penetration and toxicity by the aggregated forms. However, when applied to brain tissue sections just prior to homogenization, both monomeric and fibrillated forms stimulated glutamate release. Immuno-gold and transmission electron microscopy (TEM) detected exogenous fibrillated α-synuclein associated with numerous SN membranous structures including synaptic terminals. Western blots and immuno-gold TEM were consistent with SN internalization of α-synuclein. Additional studies revealed no evidence of gross disruption of SN membrane integrity or glutamate transporter function by exogenous α-synuclein. Overall excitotoxicity, due to enhanced glutamate release in the face of either overexpressed monomeric α-synuclein or extrasynaptic exposure to fibrillated α-synuclein, should be considered as a potential neuropathological pathway during the progression of PD and other synucleinopathies. © 2017 Wiley Periodicals, Inc.
Assuntos
Ácido Glutâmico/metabolismo , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/farmacologia , Animais , Humanos , Camundongos , Doença de ParkinsonRESUMO
Aberrant elevation in the levels of the pro-inflammatory cytokine interleukin-1ß (IL-1ß) contributes to neuroinflammatory diseases. Blood-brain barrier (BBB) dysfunction is a hallmark phenotype of neuroinflammation. It is known that IL-1ß directly induces BBB hyperpermeability but the mechanisms remain unclear. Claudin-5 (Cldn5) is a tight junction protein found at endothelial cell-cell contacts that are crucial for maintaining brain microvascular endothelial cell (BMVEC) integrity. Transcriptional regulation of Cldn5 has been attributed to the transcription factors ß-catenin and forkhead box protein O1 (FoxO1), and the signaling molecules regulating their nuclear translocation. Non-muscle myosin light chain kinase (nmMlck, encoded by the Mylk gene) is a key regulator involved in endothelial hyperpermeability, and IL-1ß has been shown to mediate nmMlck-dependent barrier dysfunction in epithelia. Considering these factors, we tested the hypothesis that nmMlck modulates IL-1ß-mediated downregulation of Cldn5 in BMVECs in a manner that depends on transcriptional repression mediated by ß-catenin and FoxO1. We found that treating BMVECs with IL-1ß induced barrier dysfunction concomitantly with the nuclear translocation of ß-catenin and FoxO1 and the repression of Cldn5. Most importantly, using primary BMVECs isolated from mice null for nmMlck, we identified that Cldn5 repression caused by ß-catenin and FoxO1 in IL-1ß-mediated barrier dysfunction was dependent on nmMlck.
Assuntos
Barreira Hematoencefálica/fisiopatologia , Claudina-5/genética , Células Endoteliais/fisiologia , Fatores de Transcrição Forkhead/fisiologia , Interleucina-1beta/fisiologia , Quinase de Cadeia Leve de Miosina/fisiologia , beta Catenina/fisiologia , Animais , Antígenos CD/metabolismo , Encéfalo/irrigação sanguínea , Caderinas/metabolismo , Células Cultivadas , Claudina-5/metabolismo , Regulação para Baixo , Endotélio Vascular/fisiopatologia , Proteína Forkhead Box O1 , Camundongos , Microvasos/patologia , Sequências Reguladoras de Ácido Nucleico , Transdução de Sinais , Ativação TranscricionalRESUMO
BACKGROUND: Loss of critical endothelial cell function and subsequent vascular smooth muscle cell (VSMC) migration is central to the pathology of injury-induced neointimal hyperplasia and recurrent stenosis. Thrombomodulin (TM), well known for its function as an endothelial surface anticoagulant, may have an unknown direct effect on VSMC physiology that would be lost after injury. Here, we examined a novel effect of TM on VSMC by testing the hypothesis that direct application of TM induces favorable changes to the morphology of VSMC and inhibits their migration. METHODS: Primary human VSMC were harvested using the explant technique and used in early passage (1-4) for all experiments. Laser-scanning confocal fluorescent imaging was performed to assess the effect of soluble TM on VSMC morphology. In vitro, migration of VSMC was measured using: (1) a 4-hr modified Boyden chemotaxis assay and (2) a 24-hr electric cell-substrate impedance sensing injury migration assay. Migration experiments were conducted with VSMC exposed to increasing doses of soluble recombinant TM. Recombinant thrombin served as a positive control and serum-free media as a negative control for all experimentation. Data were analyzed using a Student's t-test or repeated measures analysis of variance where appropriate (α < 0.05). RESULTS: VSMC exposed to TM clearly demonstrated a quiescent morphology with organized stress fibers consistent with a quiescent, differentiated, contractile phenotype; whereas, thrombin stimulation led to an activated, dedifferentiated, synthetic phenotype. VSMC demonstrated a low, baseline level of migration in unstimulated serum-free conditions. Thrombin significantly stimulated VSMC migration as expected. TM, independent of thrombin, significantly inhibited baseline VSMC migration in a dose-response fashion. The maximal inhibition was observed at (5 µg/mL) with 70% reduction (56 ± 1.7 vs. 18 ± 3.5 cells/5 high-power fields, P = 0.0005). CONCLUSIONS: TM has a direct effect on VSMC resulting in a quiescent, differentiated and contractile phenotype, and inhibition of migration. This effect is independent of the presence of thrombin. These findings provide new knowledge in understanding the pathophysiology of vascular injury and support a strategy focused on restoring key endothelial function to prevent intimal hyperplasia.
Assuntos
Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Trombomodulina , Técnicas de Cultura de Células , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/patologia , Miócitos de Músculo Liso/fisiologia , Fenótipo , TrombinaRESUMO
The microvascular endothelium has a critical role in regulating the delivery of oxygen, nutrients, and water to the surrounding tissues. Under inflammatory conditions that accompany acute injury or disease, microvascular permeability becomes elevated. When microvascular hyperpermeability becomes uncontrolled or chronic, the excessive escape of plasma proteins into the surrounding tissue disrupts homeostasis and ultimately leads to organ dysfunction. Much remains to be learned about the mechanisms that control microvascular permeability. In addition to in vivo and isolated microvessel methods, the cultured endothelial cell monolayer protocol is an important tool that allows for understanding the specific, endothelial subcellular mechanisms that determine permeability of the endothelium to plasma proteins. In this chapter, two variations of the popular Transwell culture methodology to determine permeability to using fluorescently labeled tracers are presented. The strengths and weaknesses of this approach are also discussed.
Assuntos
Permeabilidade Capilar , Células Endoteliais , Células Endoteliais/metabolismo , Endotélio/metabolismo , Permeabilidade Capilar/fisiologia , Células Cultivadas , Proteínas Sanguíneas/metabolismo , Permeabilidade , Endotélio Vascular/metabolismoRESUMO
The COVID pandemic exposed the vulnerability of older adults in myriad ways and social service organizations faced unprecedented challenges in safely providing support for older adults. Since 2007, Hawai'i Healthy Aging Partnership (HHAP) has offered Enhance®Fitness, an evidence-based program to reduce the risk of falls and promote health among older adults. Due to the pandemic, all the Enhance®Fitness sites had to close and stop offering the program. The HHAP started to provide alternative activities remotely in May 2020. To explore the pandemic's impact, the feasibility of online exercise programs, and the support needed among older adults to stay physically active, HHAP surveyed existing Enhance®Fitness participants and received 291 responses (59% response rate). The study used frequency distributions, comparison of means, and chi-square to analyze the survey data. Findings showed that the shutdown of the group exercise program during the pandemic led to a health status decline, a reduction in physical activities, and a shift from group to individual physical activities among older adult participants. Most respondents tried the remote exercise opportunities during the pandemic and would consider joining the remote programs in the future. However, about one-fourth of the respondents did not participate in remote exercise activities due to the lack of electronic devices, internet access, or interest in remote activity formats. To ensure equitable access to physical exercise programs for older adults in the post-pandemic era, it is critical to address the access challenges and resources needed for providing multiple programming options.
Assuntos
COVID-19 , Exercício Físico , Humanos , COVID-19/epidemiologia , COVID-19/prevenção & controle , Idoso , Masculino , Feminino , Havaí/epidemiologia , SARS-CoV-2 , Pandemias , Promoção da Saúde/métodos , Idoso de 80 Anos ou mais , Pessoa de Meia-Idade , Terapia por Exercício/métodos , Inquéritos e Questionários , Envelhecimento SaudávelRESUMO
ABSTRACT: Severe burns are associated with massive tissue destruction and cell death where nucleus histones and other damage-associated molecular patterns are released into the circulation and contribute to the pathogenesis of multiple-organ dysfunction. Currently, there is limited information regarding the pathophysiology of extracellular histones after burns, and the mechanisms underlying histone-induced vascular injury are not fully understood. In this study, by comparing the blood samples from healthy donors and burn patients, we confirmed that burn injury promoted the release of extracellular histones into the circulation, evidenced by increased plasma levels of histones correlating with injury severity. The direct effects of extracellular histones on human endothelial monolayers were examined, and the results showed that histones caused cell-cell adherens junction discontinuity and barrier dysfunction in a dose-related manner. Like burn patients, mice subjected to a scald burn covering 25% total body surface area also displayed significantly increased plasma histones. Intravital microscopic analysis of mouse mesenteric microcirculation indicated that treatment with a histone antibody greatly attenuated burn-induced plasma leakage in postcapillary venules, supporting the pathogenic role of extracellular histones in the development of microvascular barrier dysfunction during burns. At the molecular level, intrigued by the recent discovery of C-type lectin domain family 2 member D (Clec2d) as a novel receptor of histones, we tested its potential involvement in the histone interaction with endothelial cells. Indeed, we identified abundant expression of Clec2d in vascular endothelial cells. Further proximity ligation assay demonstrated a close association between extracellular histones and endothelial expressing Clec2d. Functionally, in vivo administration of an anti-Clec2d antibody attenuated burn-induced plasma leakage across mesenteric microvessels. Consistently, Clec2d knockdown in endothelial cells partially inhibited histone-induced endothelial barrier dysfunction. Together, our data suggest that burn injury-induced increases in circulating histones contribute to microvascular leakage and endothelial barrier dysfunction via a mechanism involving the endothelial Clec2d receptor.
Assuntos
Queimaduras , Histonas , Humanos , Camundongos , Animais , Histonas/metabolismo , Células Endoteliais/metabolismo , Lectinas Tipo C/metabolismo , Endotélio Vascular/patologia , Queimaduras/patologiaRESUMO
Microvascular barrier dysfunction is a serious problem that occurs in many inflammatory conditions, including sepsis, trauma, ischemia-reperfusion injury, cardiovascular disease, and diabetes. Barrier dysfunction permits extravasation of serum components into the surrounding tissue, leading to edema formation and organ failure. The basis for microvascular barrier dysfunction is hyperpermeability at endothelial cell-cell junctions. Endothelial hyperpermeability is increased by actomyosin contractile activity in response to phosphorylation of myosin light chain by myosin light chain kinase (MLCK). MLCK-dependent endothelial hyperpermeability occurs in response to inflammatory mediators (e.g., activated neutrophils, thrombin, histamine, tumor necrosis factor alpha, etc.), through multiple cell signaling pathways and signaling molecules (e.g., Ca(++) , protein kinase C, Src kinase, nitric oxide synthase, etc.). Other signaling molecules protect against MLCK-dependent hyperpermeability (e.g., sphingosine-1-phosphate or cAMP). In addition, individual MLCK isoforms play specific roles in endothelial barrier dysfunction, suggesting that isoform-specific inhibitors could be useful for treating inflammatory disorders and preventing multiple organ failure. Because endothelial barrier dysfunction depends upon signaling through MLCK in many instances, MLCK-dependent signaling comprises multiple potential therapeutic targets for preventing edema formation and multiple organ failure. The following review is a discussion of MLCK-dependent mechanisms and cell signaling events that mediate endothelial hyperpermeability.
Assuntos
Endotélio/enzimologia , Quinase de Cadeia Leve de Miosina/metabolismo , Transdução de Sinais , Animais , Endotélio/efeitos dos fármacos , Endotélio/fisiopatologia , Humanos , Terapia de Alvo Molecular , Quinase de Cadeia Leve de Miosina/química , Permeabilidade/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
ADAM15 is a disintegrin and metalloprotease recently implicated in cancer and chronic immune disorders. We have recently characterized ADAM15 as a mediator of endothelial barrier dysfunction. Whether this molecule contributes to acute inflammation has not been evaluated. The purpose of this study was to investigate the role of ADAM15 in mediating pulmonary microvascular leakage during acute inflammatory injury. Immunofluorescent staining and Western blotting revealed that the endothelium was the main source of ADAM15 in lung tissue. In a mouse model of acute lung injury induced by lipopolysaccharide (LPS), upregulation of ADAM15 was observed in association with pulmonary edema and neutrophil infiltration. The LPS-induced inflammatory injury, as demonstrated by bronchoalveolar lavage neutrophil count, lung wet-to-dry weight ratio, and myeloperoxidase activity, was significantly attenuated in Adam15(-/-) mice. Studies with primary cell culture demonstrated abundant ADAM15 expression in endothelial cells (ECs) of mouse lung but not in neutrophils. Deficiency of ADAM15 in ECs had no obvious effect on basal permeability but significantly attenuated hyperpermeability response to LPS as evidenced by albumin flux assay and measurements of transendothelial electrical resistance, respectively. ADAM15 deficiency also reduced neutrophil chemotactic transmigration across endothelial barriers in the presence or absence of formyl-methionyl-leucyl-phenylalanine (fMLP). Rescue expression of ADAM15 in Adam15(-/-) ECs restored neutrophil transendothelial migration. These data indicate that ADAM15 upregulation contributes to inflammatory lung injury by promoting endothelial hyperpermeability and neutrophil transmigration.
Assuntos
Proteínas ADAM/genética , Lesão Pulmonar Aguda/metabolismo , Células Endoteliais/metabolismo , Pulmão/metabolismo , Proteínas de Membrana/genética , Neutrófilos/metabolismo , Edema Pulmonar/metabolismo , Proteínas ADAM/deficiência , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/patologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Impedância Elétrica , Células Endoteliais/patologia , Lipopolissacarídeos/farmacologia , Pulmão/patologia , Proteínas de Membrana/deficiência , Camundongos , Camundongos Knockout , Infiltração de Neutrófilos , Neutrófilos/patologia , Permeabilidade , Peroxidase/genética , Peroxidase/metabolismo , Cultura Primária de Células , Edema Pulmonar/induzido quimicamente , Edema Pulmonar/genética , Edema Pulmonar/patologia , Migração Transendotelial e Transepitelial , Regulação para CimaRESUMO
OBJECTIVE: Endothelium dysfunction is an initiating factor in atherosclerosis. A disintegrin and metalloproteinase 15 (ADAM 15) is a multidomain metalloprotease recently identified as a regulator of endothelial permeability. However, whether and how ADAM15 contributes to atherosclerosis remains unknown. METHODS AND RESULTS: Genetic ablation of ADAM15 in apolipoprotein E-deficient mice led to a significant reduction in aortic atherosclerotic lesion size (by 52%), plaque macrophage infiltration (by 69%), and smooth muscle cell deposition (by 82%). In vitro studies implicated endothelial-derived ADAM15 in barrier dysfunction and monocyte transmigration across mouse aortic and human umbilical vein endothelial cell monolayers. This role of ADAM15 depended on intact functioning of the cytoplasmic domain, as evidenced in experiments with site-directed mutagenesis targeting the metalloprotease active site (E349A), the disintegrin domain (Arginine-Glycine-Aspartic acidâThreonine-Aspartic acid-Aspartic acid), or the cytoplasmic tail. Further investigations revealed that ADAM15-induced barrier dysfunction was concomitant with dissociation of endothelial adherens junctions (vascular endothelial [VE]-cadherin/γ-catenin), an effect that was sensitive to Src family kinase inhibition. Through small interfering RNA-mediated knockdown of distinct Src family kinase members, c-Src and c-Yes were identified as important mediators of these junctional effects of ADAM15. CONCLUSIONS: These results suggest that endothelial cell-derived ADAM15, signaling through c-Src and c-Yes, contributes to atherosclerotic lesion development by disrupting adherens junction integrity and promoting monocyte transmigration.