RESUMEN
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.
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Linfopenia , Infarto del Miocardio , Animales , Médula Ósea , Humanos , Recuento de Linfocitos , Linfocitos , Linfopenia/inducido químicamente , Ratones , Infarto del Miocardio/complicacionesRESUMEN
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.
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Tracto Gastrointestinal/patología , Pulmón/patología , Vasos Linfáticos/patología , Síndrome de Respuesta Inflamatoria Sistémica/patología , Lesión Pulmonar Aguda/patología , Animales , HumanosRESUMEN
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.
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Permeabilidad Capilar , Endotelio Vascular/metabolismo , Vesículas Extracelulares/metabolismo , Mediadores de Inflamación/metabolismo , Inflamación/metabolismo , Animales , Betacoronavirus/patogenicidad , COVID-19 , Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Endotelio Vascular/patología , Endotelio Vascular/virología , Vesículas Extracelulares/patología , Vesículas Extracelulares/virología , Interacciones Huésped-Patógeno , Humanos , Inflamación/patología , Pandemias , Neumonía Viral/metabolismo , Neumonía Viral/patología , Neumonía Viral/virología , SARS-CoV-2 , Transducción de SeñalRESUMEN
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.
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Histonas/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Animales , Células Cultivadas , Colforsina/farmacología , Histonas/sangre , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
hsc-70 (HSPA8) is a cytosolic molecular chaperone, which plays a central role in cellular proteostasis, including quality control during protein refolding and regulation of protein degradation. hsc-70 is pivotal to the process of macroautophagy, chaperone-mediated autophagy, and endosomal microautophagy. The latter requires hsc-70 interaction with negatively charged phosphatidylserine (PS) at the endosomal limiting membrane. Herein, by combining plasmon resonance, NMR spectroscopy, and amino acid mutagenesis, we mapped the C terminus of the hsc-70 LID domain as the structural interface interacting with endosomal PS, and we estimated an hsc-70/PS equilibrium dissociation constant of 4.7 ± 0.1 µm. This interaction is specific and involves a total of 4-5 lysine residues. Plasmon resonance and NMR results were further experimentally validated by hsc-70 endosomal binding experiments and endosomal microautophagy assays. The discovery of this previously unknown contact surface for hsc-70 in this work elucidates the mechanism of hsc-70 PS/membrane interaction for cytosolic cargo internalization into endosomes.
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Autofagia/fisiología , Endosomas/metabolismo , Proteínas del Choque Térmico HSC70/metabolismo , Membranas Intracelulares/metabolismo , Fosfatidilserinas/metabolismo , Animales , Línea Celular , Endosomas/química , Endosomas/genética , Proteínas del Choque Térmico HSC70/química , Proteínas del Choque Térmico HSC70/genética , Membranas Intracelulares/química , Ratones , Fosfatidilserinas/química , Fosfatidilserinas/genéticaRESUMEN
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.
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Barrera Hematoencefálica/fisiopatología , Claudina-5/genética , Células Endoteliales/fisiología , Factores de Transcripción Forkhead/fisiología , Interleucina-1beta/fisiología , Quinasa de Cadena Ligera de Miosina/fisiología , beta Catenina/fisiología , Animales , Antígenos CD/metabolismo , Encéfalo/irrigación sanguínea , Cadherinas/metabolismo , Células Cultivadas , Claudina-5/metabolismo , Regulación hacia Abajo , Endotelio Vascular/fisiopatología , Proteína Forkhead Box O1 , Ratones , Microvasos/patología , Secuencias Reguladoras de Ácidos Nucleicos , Transducción de Señal , Activación TranscripcionalRESUMEN
OBJECTIVES: The knowledge of the basic principles of lymphatic function, still remains, to a large degree, rudimentary and will require significant research efforts. Recent studies of the physiology of the MLVs suggested the presence of an EDRF other than NO. In this study, we tested the hypothesis that lymphatic endothelium-derived histamine relaxes MLVs. METHODS: We measured and analyzed parameters of lymphatic contractility in isolated and pressurized rat MLVs under control conditions and after pharmacological blockade of NO by L-NAME (100 µM) or/and histamine production by α-MHD (10 µM). Effectiveness of α-MHD was confirmed immunohistochemically. We also used immunohistochemical labeling and Western blot analysis of the histamine-producing enzyme, HDC. In addition, we blocked HDC protein expression in MLVs by transient transfection with vivo-morpholino oligos. RESULTS: We found that only combined pharmacological blockade of NO and histamine production completely eliminates flow-dependent relaxation of lymphatic vessels, thus confirming a role for histamine as an EDRF in MLVs. We also confirmed the presence of HDC and histamine inside lymphatic endothelial cells. CONCLUSIONS: This study supports a role for histamine as an EDRF in MLVs.
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Endotelio Linfático/fisiología , Histamina/fisiología , Vasos Linfáticos/fisiología , Óxido Nítrico/fisiología , Animales , Endotelio Linfático/citología , Endotelio Linfático/efectos de los fármacos , Guanilato Ciclasa/antagonistas & inhibidores , Guanilato Ciclasa/fisiología , Histamina/análisis , Histidina Descarboxilasa/fisiología , Vasos Linfáticos/efectos de los fármacos , Masculino , Mesenterio , Metilhistidinas/farmacología , Morfolinos/farmacología , Relajación Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/antagonistas & inhibidores , Ratas , Ratas Endogámicas F344 , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/fisiología , Guanilil Ciclasa SolubleRESUMEN
We had previously proposed the presence of permanent stimulatory influences in the tissue microenvironment surrounding the aged mesenteric lymphatic vessels (MLV), which influence aged lymphatic function. In this study, we performed immunohistochemical labeling of proteins known to be present in mast cells (mast cell tryptase, c-kit, prostaglandin D(2) synthase, histidine decarboxylase, histamine, transmembrane protein 16A, and TNF-α) with double verification of mast cells in the same segment of rat mesentery containing MLV by labeling with Alexa Fluor 488-conjugated avidin followed by toluidine blue staining. Additionally, we evaluated the aging-associated changes in the number of mast cells located by MLV and in their functional status by inducing mast cell activation by various activators (substance P; anti-rat DNP Immunoglobulin E; peptidoglycan from Staphyloccus aureus and compound 48/80) in the presence of ruthenium red followed by subsequent staining by toluidine blue. We found that there was a 27% aging-associated increase in the total number of mast cells, with an â¼400% increase in the number of activated mast cells in aged mesenteric tissue in resting conditions with diminished ability of mast cells to be newly activated in the presence of inflammatory or chemical stimuli. We conclude that higher degree of preactivation of mast cells in aged mesenteric tissue is important for development of aging-associated impairment of function of mesenteric lymphatic vessels. The limited number of intact aged mast cells located close to the mesenteric lymphatic compartments to react to the presence of acute stimuli may be considered contributory to the aging-associated deteriorations in immune response.
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Envejecimiento/inmunología , Microambiente Celular , Vasos Linfáticos/inmunología , Mastocitos/inmunología , Factores de Edad , Envejecimiento/metabolismo , Animales , Anoctamina-1 , Biomarcadores/metabolismo , Canales de Cloruro/metabolismo , Histidina Descarboxilasa/metabolismo , Inmunoglobulina E/farmacología , Inmunohistoquímica , Oxidorreductasas Intramoleculares/metabolismo , Lipocalinas/metabolismo , Vasos Linfáticos/metabolismo , Masculino , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Mesenterio , Peptidoglicano/aislamiento & purificación , Peptidoglicano/farmacología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Ratas , Ratas Endogámicas F344 , Coloración y Etiquetado , Staphylococcus aureus/química , Sustancia P/farmacología , Triptasas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , p-Metoxi-N-metilfenetilamina/farmacologíaRESUMEN
Extracellular vesicles (EVs) are bioactive membrane-encapsulated particles generated by a series of events involving membrane budding, fission and fusion. Palmitoylation, mediated by DHHC palmitoyl acyltransferases, is a lipidation reaction that increases protein lipophilicity and membrane localization. Here, we report palmitoylation as a novel regulator of EV formation and function during sepsis. Our results showed significantly decreased circulating EVs in mice with DHHC21 functional deficiency (Zdhhc21dep/dep), compared to wild-type (WT) mice 24 h after septic injury. Furthermore, WT and Zdhhc21dep/dep EVs displayed distinct palmitoyl-proteomic profiles. Ingenuity pathway analysis indicated that sepsis altered several inflammation related pathways expressed in EVs, among which the most significantly activated was the complement pathway; however, this sepsis-induced complement enrichment in EVs was greatly blunted in Zdhhc21dep/dep EVs. Functionally, EVs isolated from WT mice with sepsis promoted neutrophil adhesion, transmigration, and neutrophil extracellular trap production; these effects were significantly attenuated by DHHC21 loss-of-function. Furthermore, Zdhhc21dep/dep mice displayed reduced neutrophil infiltration in lungs and improved survival after CLP challenges. These findings indicate that blocking palmitoylation via DHHC21 functional deficiency can reduce sepsis-stimulated production of complement-enriched EVs and attenuates their effects on neutrophil activity.
RESUMEN
ABSTRACT: Extracellular vesicles (EVs) are nano-sized membrane-bound particles containing biologically active cargo molecules. The production and molecular composition of EVs reflect the physiological state of parent cells, and once released into the circulation, they exert pleiotropic functions via transferring cargo contents. Thus, circulating EVs not only serve as biomarkers, but also mediators in disease processes or injury responses. In the present study, we performed a comprehensive analysis of plasma EVs from burn patients and healthy subjects, characterizing their size distribution, concentration, temporal changes, cell origins, and cargo protein contents. Our results indicated that burn injury induced a significant increase in circulating EVs, the response peaked at the time of admission and declined over the course of recovery. Importantly, EV production correlated with injury severity, as indicated by the total body surface area and depth of burn, requirement for critical care/ICU stay, hospitalization length, wound infection, and concurrence of sepsis. Burn patients with inhalation injury showed a higher level of EVs than those without inhalation injury. We also evaluated patient demographics (age and sex) and pre-existing conditions (hypertension, obesity, and smoking) and found no significant correlation between these conditions and overall EV production. At the molecular level, flow cytometric analysis showed that the burn-induced EVs were largely derived from leukocytes and endothelial cells (ECs), which are known to be activated postburn. Additionally, a high level of zona-occludens-1 (ZO-1), a major constituent of tight junctions, was identified in burn EV cargos, indicative of injury in tissues that form barriers via tight junctions. Moreover, when applied to endothelial cell monolayers, burn EVs caused significant barrier dysfunction, characterized by decreased transcellular barrier resistance and disrupted cell-cell junction continuity. Taken together, these data suggest that burn injury promotes the production of EVs containing unique cargo proteins in a time-dependent manner; the response correlates with injury severity and worsened clinical outcomes. Functionally, burn EVs serve as a potent mediator capable of reducing endothelial barrier resistance and impairing junction integrity, a pathophysiological process underlying burn-associated tissue dysfunction. Thus, further in-depth characterization of circulating EVs will contribute to the development of new prognostic tools or therapeutic targets for advanced burn care.
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Quemaduras , Vesículas Extracelulares , Quemaduras/complicaciones , Quemaduras/metabolismo , Comunicación Celular , Células Endoteliales/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Uniones EstrechasRESUMEN
Renal dysfunction is one of the most common complications of septic injury. One critical contributor to septic injury-induced renal dysfunction is renal vascular dysfunction. Protein palmitoylation serves as a novel regulator of vascular function. Here, we examined whether palmitoyl acyltransferase (PAT)-DHHC21 contributes to septic injury-induced renal dysfunction through regulating renal hemodynamics. Multispectral optoacoustic imaging showed that cecal ligation and puncture (CLP)-induced septic injury caused impaired renal excretion, which was improved in DHHC21 functional deficient (Zdhhc21dep/dep) mice. DHHC21 deficiency attenuated CLP-induced renal pathology, characterized by tissue structural damage and circulating injury markers. Importantly, DHHC21 loss-of-function led to better-preserved renal perfusion and oxygen saturation after CLP. The CLP-caused reduction in renal blood flow was also ameliorated in Zdhhc21dep/dep mice. Next, CLP promoted the palmitoylation of vascular α1-adrenergic receptor (α1AR) and the activation of its downstream effector ERK, which were blunted in Zdhhc21dep/dep mice. Vasoreactivity analysis revealed that renal arteries from Zdhhc21dep/dep mice displayed reduced constriction response to α1AR agonist phenylephrine compared to those from wild-type mice. Consistently, inhibiting PATs with 2-bromopalmitate caused a blunted vasoconstriction response to phenylephrine in small arteries isolated from human kidneys. Therefore, DHHC21 contributes to impaired renal perfusion and function during septic injury via promoting α1AR palmitoylation-associated vasoconstriction.
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Aciltransferasas/genética , Enfermedades Renales/fisiopatología , Riñón/fisiopatología , Sepsis/fisiopatología , Animales , Ciego/metabolismo , Ciego/fisiopatología , Riñón/metabolismo , Enfermedades Renales/etiología , Enfermedades Renales/genética , Lipoilación , Ratones , Ratones Noqueados , Receptores Adrenérgicos alfa 1/metabolismo , Sepsis/complicaciones , Sepsis/genéticaRESUMEN
Extracellular vesicles (EV) have been implicated in diverse biological processes, including intracellular communication, transport of nucleic acids, and regulation of vascular function. Levels of EVs are elevated in cancer, and studies suggest that EV may stimulate thrombosis in patients with cancer through expression of tissue factor. However, limited data also implicate EV in the activation of the contact pathway of coagulation through activation of factor XII (FXII) to FXIIa. To better define the ability of EV to initiate contact activation, we compared the ability of EV derived from different cancer cell lines to activate FXII. EV from all cell lines activated FXII, with those derived from pancreatic and lung cancer cell lines demonstrating the most potent activity. Concordant with the activation of FXII, EV induced the cleavage of high molecular weight kininogen (HK) to cleaved kininogen. We also observed that EVs from patients with cancer stimulated FXII activation and HK cleavage. To define the mechanisms of FXII activation by EV, EV were treated with calf intestinal alkaline phosphatase or Escherichia coli exopolyphosphatase to degrade polyphosphate; this treatment blocked binding of FXII to EVs and the ability of EV to mediate FXII activation. In vivo, EV induced pulmonary thrombosis in wild-type mice, with protection conferred by a deficiency in FXII, HK, or prekallikrein. Moreover, pretreatment of EVs with calf intestinal alkaline phosphatase inhibited their prothrombotic effect. These results indicate that polyphosphate mediates the binding of contact factors to EV and that EV-associated polyphosphate may contribute to the prothrombotic effects of EV in cancer.
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Vesículas Extracelulares , Neoplasias , Animales , Factor XII , Factor XIIa , Humanos , Ratones , Polifosfatos , PrecalicreínaRESUMEN
Palmitoylation is a post-translational modification (PTM) based on thioester-linkage between palmitic acid and the cysteine residue of a protein. This covalent attachment of palmitate is reversibly and dynamically regulated by two opposing sets of enzymes: palmitoyl acyltransferases containing a zinc finger aspartate-histidine-histidine-cysteine motif (PAT-DHHCs) and thioesterases. The reversible nature of palmitoylation enables fine-tuned regulation of protein conformation, stability, and ability to interact with other proteins. More importantly, the proper function of many surface receptors and signaling proteins requires palmitoylation-meditated partitioning into lipid rafts. A growing number of leukocyte proteins have been reported to undergo palmitoylation, including cytokine/chemokine receptors, adhesion molecules, pattern recognition receptors, scavenger receptors, T cell co-receptors, transmembrane adaptor proteins, and signaling effectors including the Src family of protein kinases. This review provides the latest findings of palmitoylated proteins in leukocytes and focuses on the functional impact of palmitoylation in leukocyte function related to adhesion, transmigration, chemotaxis, phagocytosis, pathogen recognition, signaling activation, cytotoxicity, and cytokine production.
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Gut ischemia/reperfusion (I/R) injury is a common clinical problem associated with significant mortality and morbidities that result from systemic inflammation and remote organ dysfunction, typically acute lung injury. The mechanisms underlying the dissemination of gut-derived harmful mediators into the circulation are poorly understood. The objective of our study was to determine the role of mesenteric lymphatic circulation in the systemic and pulmonary inflammatory response to gut I/R. Using a murine intestinal I/R model, we evaluated whether and how blocking mesenteric lymph flow affects the inflammatory response in local tissues (gut) and remote organs (lungs). We further explored the mechanisms of post-I/R lymph-induced systemic inflammation by examining neutrophil activity and interaction with endothelial cells in vitro. Mice subjected to intestinal I/R displayed a significant inflammatory response in local tissues, evidenced by neutrophil infiltration into mucosal areas, as well as lung inflammation, evidenced by increased myeloperoxidase levels, neutrophil infiltration, and elevated microvascular permeability in the lungs. Mesenteric lymph duct ligation (MLDL) had no effect on gut injury per se, but effectively attenuated lung injury following gut I/R. Cell experiments showed that lymph fluid from post-I/R animals, but not pre-I/R, increased neutrophil surface CD11b expression and their ability to migrate across vascular endothelial monolayers. Moreover, post-I/R lymph upregulated neutrophil expression of pro-inflammatory cytokines and chemokines, which was mediated by a mechanism involving nuclear factor (NF)-κB signaling. Consistently, gut I/R activated NF-κB in lung neutrophils, which was alleviated by MLDL. In conclusion, all these data indicate that mesenteric lymph circulation contributes to neutrophil activation and lung inflammation following gut I/R injury partly through activating NF-κB.
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Sistema Linfático/inmunología , Activación Neutrófila/inmunología , Neumonía/inmunología , Daño por Reperfusión/inmunología , Animales , Intestinos/inmunología , Intestinos/lesiones , Intestinos/patología , Masculino , Mesenterio/inmunología , Ratones , Ratones Endogámicos C57BL , Neumonía/metabolismo , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismoRESUMEN
AIMS: Microvesicles (MVs) conduct intercellular communication and impact diverse biological processes by transferring bioactive cargos to other cells. We investigated whether and how endothelial production of MVs contribute to vascular dysfunction during inflammation. METHODS AND RESULTS: We measured the levels and molecular properties of endothelial-derived MVs (EC-MVs) from mouse plasma following a septic injury elicited by cecal ligation and puncture, as well as those from supernatants of cultured endothelial cells stimulated by inflammatory agents including cytokines, thrombin, and complement 5a. The mouse studies showed that sepsis caused a significant increase in total plasma vesicles and VE-cadherin+ EC-MVs compared to sham control. In cultured ECs, different inflammatory agents caused diverse patterns of EC-MV production and cargo contents. When topically applied to endothelial cells, EC-MVs induced a cytoskeleton-junction response characterized by myosin light chain phosphorylation, contractile fibre reorganization, VE-cadherin phosphorylation, and adherens junction dissociation, functionally measured as increased albumin transendothelial flux and decreased barrier resistance. The endothelial response was coupled with protein tyrosine phosphorylation promoted by MV cargo containing c-Src kinase, whereas MVs produced from c-Src deficient cells did not exert barrier-disrupting effects. Additionally, EC-MVs contribute to endothelial inflammatory injury by promoting neutrophil-endothelium adhesion and release of neutrophil extracellular traps containing citrullinated histones and myeloperoxidase, a response unaltered by c-Src knockdown. CONCLUSION: Endothelial-derived microparticles cause endothelial barrier dysfunction by impairing adherens junctions and activating neutrophils. The signalling mechanisms underlying the endothelial cytoskeleton-junction response to EC-MVs involve protein phosphorylation promoted by MV cargo carrying c-Src. However, EC-MV-induced neutrophil activation was not dependent on c-Src.
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Uniones Adherentes/metabolismo , Micropartículas Derivadas de Células/enzimología , Citoesqueleto/metabolismo , Células Endoteliales/enzimología , Células Endoteliales de la Vena Umbilical Humana/enzimología , Inflamación/enzimología , Sepsis/enzimología , Familia-src Quinasas/metabolismo , Uniones Adherentes/patología , Adolescente , Adulto , Animales , Micropartículas Derivadas de Células/patología , Células Cultivadas , Citoesqueleto/patología , Modelos Animales de Enfermedad , Células Endoteliales/patología , Femenino , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Inflamación/patología , Mediadores de Inflamación/metabolismo , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Permeabilidad , Transporte de Proteínas , Sepsis/patología , Adulto JovenRESUMEN
The microvascular endothelium serves as the major barrier that controls the transport of blood constituents across the vessel wall. Barrier leakage occurs during infection or sterile inflammation, allowing plasma fluid and cells to extravasate and accumulate in surrounding tissues, an important pathology underlying a variety of infectious diseases and immune disorders. The leak process is triggered and regulated by bidirectional communications between circulating cells and vascular cells at the blood-vessel interface. While the molecular mechanisms underlying this complex process remain incompletely understood, emerging evidence supports the roles of neutrophil-endothelium interaction and neutrophil-derived products, including neutrophil extracellular traps and vesicles, in the pathogenesis of vascular barrier injury. In this review, we summarize the current knowledge on neutrophil-induced changes in endothelial barrier structures, with a detailed presentation of recently characterized molecular pathways involved in the production and effects of neutrophil extracellular traps and extracellular vesicles. Additionally, we discuss the therapeutic implications of altering neutrophil interactions with the endothelial barrier in treating inflammatory diseases.
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Endotelio Vascular/patología , Trampas Extracelulares/inmunología , Vesículas Extracelulares/metabolismo , Inflamación/inmunología , Neutrófilos/inmunología , Animales , Permeabilidad Capilar , Endotelio Vascular/metabolismo , HumanosRESUMEN
BACKGROUND: Aging impairs mesenteric lymph flow, which is crucial for fluid and macromolecule homeostasis, fat absorption, and immune function. Previously, we demonstrated that mast cells (MCs) line mesenteric lymphatic vessels (MLVs) with a greater degree of basal activation of MCs in aged mesentery. The number of intact MCs available to react acutely to inflammatory stimuli was decreased with age. However, the role of mast cells in recruiting other immune cells towards MLVs and its aging-associated alterations has not been explored before in great detail. METHODS AND RESULTS: In this study we treated live mesenteric tissue isolated from Sprague Dawley (SD) rats, as well as adult 9-mo and aged 24-mo Fischer-344 (F-344) rats for 2 hours with MC activators (48/80 and Substance P) and performed whole mount IHC and vital dye staining of the mesenteric segments containing MLVs to identify immune cell recruitment towards MLVs after mast cell (MC) activation. Number of major histocompatibility complex (MHC) class II positive APCs and eosinophils near MLVs was counted and compared between treatments and ages. CONCLUSIONS: With greater density of MCs near MLVs, we for the first time demonstrated that mesenteric MC activation by compound 48/80 and Substance P resulted in recruitment of MHC class II positive cells and eosinophils towards MLVs. This effect was reduced in cromolyn-injected rats, thus confirming that MCs are necessary for such recruitment. The immune cell presence near MLVs after MC activation was reduced in aged tissues. We link these findings to our previous report of lesser number of intact MCs available for initiating an acute immune response in aged mesentery. Cumulatively, these findings serve as the first step in study of the aging-associated mechanisms that link MCs, lymphatic vessels, and disordered immune function in the elderly.
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Envejecimiento/inmunología , Eosinófilos/inmunología , Vasos Linfáticos/inmunología , Mastocitos/inmunología , Mesenterio/inmunología , Animales , Quimiotaxis de Leucocito/inmunología , Eosinófilos/metabolismo , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Inmunohistoquímica , Vasos Linfáticos/citología , Masculino , Mastocitos/metabolismo , Mesenterio/citología , Ratas , Ratas Endogámicas F344 , Ratas Sprague-DawleyRESUMEN
A disintegrin and metalloproteinase15 (ADAM15) has been shown to be upregulated and mediate endothelial hyperpermeability during inflammation and sepsis. This molecule contains multiple functional domains with the ability to modulate diverse cellular processes including cell adhesion, extracellular matrix degradation, and ectodomain shedding of transmembrane proteins. These characteristics make ADAM15 an attractive therapeutic target in various diseases. The lack of pharmacological inhibitors specific to ADAM15 prompted our efforts to identify biological or molecular tools to alter its expression for further studying its function and therapeutic implications. The goal of this study was to determine if ADAM15-targeting microRNAs altered ADAM15-induced endothelial barrier dysfunction during septic challenge by bacterial lipopolysaccharide (LPS). An in silico analysis followed by luciferase reporter assay in human vascular endothelial cells identified miR-147b with the ability to target the 3' UTR of ADAM15. Transfection with a miR-147b mimic led to decreased total, as well as cell surface expression of ADAM15 in endothelial cells, while miR-147b antagomir produced an opposite effect. Functionally, LPS-induced endothelial barrier dysfunction, evidenced by a reduction in transendothelial electric resistance and increase in albumin flux across endothelial monolayers, was attenuated in cells treated with miR-147b mimics. In contrast, miR-147b antagomir exerted a permeability-increasing effect in vascular endothelial cells similar to that caused by LPS. Taken together, these data suggest the potential role of miR147b in regulating endothelial barrier function by targeting ADAM15 expression.
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Proteínas ADAM/genética , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica , Proteínas de la Membrana/genética , MicroARNs/genética , Interferencia de ARN , Regiones no Traducidas 3' , Proteínas ADAM/química , Proteínas ADAM/metabolismo , Secuencia de Bases , Sitios de Unión , Barrera Alveolocapilar/metabolismo , Membrana Celular/metabolismo , Regulación hacia Abajo , Células Endoteliales/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inmunofenotipificación , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , MicroARNs/química , PermeabilidadRESUMEN
Abstract An overview is presented of recent findings related to biology of aging of the lymph transport system. The authors discuss recently obtained data on the aging-associated alterations of lymphatic contractility in thoracic duct and mesenteric lymphatic vessels; on comparisons of function of aged mesenteric lymphatic vessels in situ versus isolated specimens and important conclusions which arose from these studies; on aging-associated changes in functional status of mast cells located close to aged mesenteric lymphatic vessels; on evidence of presence of oxidative stress in aged lymphatic vessels and changes in arrangement of muscle cells in their walls. The authors conclude that future continuation of the research efforts in this area is necessary and will be able to provide not only novel fundamental knowledge on the biology of lymphatic aging, but also will create solid foundation for the subsequent developments of lymphatic-oriented therapeutic interventions in many diseases of the elderly.
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Envejecimiento/fisiología , Sistema Linfático/fisiología , Músculo Liso/fisiología , Conducto Torácico/fisiología , Factores de Edad , Animales , Humanos , Mastocitos/fisiología , Mesenterio/citología , Mesenterio/fisiología , Contracción Muscular/fisiología , Músculo Liso/citologíaRESUMEN
BACKGROUND: Recent studies on aging-associated changes in mesenteric lymph flow in situ demonstrated predominance of the severe negative chronotropic effect of aging on the contractility of aged mesenteric lymphatic vessels (MLV). At the same time, contraction amplitude of the aged vessels was only slightly diminished by aging and can be rapidly stimulated within 5-15 minutes. However, the detailed quantitative evaluation of potential aging-associated changes in muscle cells investiture in MLV has never been performed. METHODS AND RESULTS: In this study we, for the first time, performed detailed evaluation of muscle cells investiture in MLV in reference to the position of lymphatic valve in different zones of lymphangion within various age groups (3-mo, 9-mo and 24-mo Fischer-344 rats). Using visual and quantitative analyses of the images of MLV immunohistochemically labeled for actin, we confirmed that the zones located close upstream (pre-valve zones) and above lymphatic valves (valve zones) possess the lowest investiture of lymphatic muscle cells. Most of the high muscle cells investiture zones exist downstream to the lymphatic valve (post-valve zones). The muscle cells investiture of these zones is not affected by aging, while pre-valve and valve zones demonstrate significant aging-associated decrease in muscle cells investiture. CONCLUSIONS: The low muscle cells investiture zones in lymphatic vessels consist of predominantly longitudinally oriented muscle cells which are positioned in pre-valve and valve zones and connect adjacent lymphangions. These cells may provide important functional impact on the biomechanics of the lymphatic valve gating and electrical coupling between lymphangions, while their aging-associated changes may delimit adaptive reserves of aged lymphatic vessels.