RESUMEN
TGF-ß-activated kinase 1 (TAK1) plays crucial roles in innate and adaptive immune responses and is required for embryonic vascular development. However, TAK1's role in regulating vascular barrier integrity is not well defined. Here we show that endothelial TAK1 kinase function is required to maintain and repair the injured lung endothelial barrier. We observed that inhibition of TAK1 with 5Z-7-oxozeaenol markedly reduced expression of ß-catenin (ß-cat) and VE-cadherin at endothelial adherens junctions and augmented protease-activated receptor-1 (PAR-1)- or toll-like receptor-4 (TLR-4)-induced increases in lung vascular permeability. In inducible endothelial cell (EC)-restricted TAK1 knockout (TAK1i∆EC) mice, we observed that the lung endothelial barrier was compromised and in addition, TAK1i∆EC mice exhibited heightened sensitivity to septic shock. Consistent with these findings, we observed dramatically reduced ß-cat expression in lung ECs of TAK1i∆EC mice. Further, either inhibition or knockdown of TAK1 blocked PAR-1- or TLR-4-induced inactivation of glycogen synthase kinase 3ß (GSK3ß), which in turn increased phosphorylation, ubiquitylation, and degradation of ß-cat in ECs to destabilize the endothelial barrier. Importantly, we showed that TAK1 inactivates GSK3ß through AKT activation in ECs. Thus our findings in this study point to the potential of targeting the TAK1-AKT-GSK3ß axis as a therapeutic approach to treat uncontrolled lung vascular leak during sepsis.
Asunto(s)
Receptor Toll-Like 4 , Lesiones del Sistema Vascular , Animales , Glucógeno Sintasa Quinasa 3 beta , Pulmón , Quinasas Quinasa Quinasa PAM , Ratones , Proteínas Proto-Oncogénicas c-aktRESUMEN
Caveolae, the cave-like structures abundant in endothelial cells (ECs), are important for multiple signaling processes such as production of nitric oxide and caveolae-mediated intracellular trafficking. Using superresolution microscopy, fluorescence resonance energy transfer, and biochemical analysis, we observed that the EphB1 receptor tyrosine kinase constitutively interacts with caveolin-1 (Cav-1), the key structural protein of caveolae. Activation of EphB1 with its ligand Ephrin B1 induced EphB1 phosphorylation and the uncoupling EphB1 from Cav-1 and thereby promoted phosphorylation of Cav-1 by Src. Deletion of Cav-1 scaffold domain binding (CSD) motif in EphB1 prevented EphB1 binding to Cav-1 as well as Src-dependent Cav-1 phosphorylation, indicating the importance of CSD in the interaction. We also observed that Cav-1 protein expression and caveolae numbers were markedly reduced in ECs from EphB1-deficient (EphB1-/-) mice. The loss of EphB1 binding to Cav-1 promoted Cav-1 ubiquitination and degradation, and hence the loss of Cav-1 was responsible for reducing the caveolae numbers. These studies identify the crucial role of EphB1/Cav-1 interaction in the biogenesis of caveolae and in coordinating the signaling function of Cav-1 in ECs.
Asunto(s)
Caveolas/metabolismo , Receptor EphB1/metabolismo , Animales , Caveolas/fisiología , Caveolina 1/metabolismo , Células Endoteliales/metabolismo , Efrina-B1/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/metabolismo , Fosforilación , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor EphB1/fisiología , Transducción de Señal/fisiologíaRESUMEN
Increased permeability of vascular lung tissues is a hallmark of acute lung injury and is often caused by edemagenic insults resulting in inflammation. Vascular endothelial (VE)-cadherin undergoes internalization in response to inflammatory stimuli and is recycled at cell adhesion junctions during endothelial barrier re-establishment. Here, we hypothesized that phospholipase D (PLD)-generated phosphatidic acid (PA) signaling regulates VE-cadherin recycling and promotes endothelial barrier recovery by dephosphorylating VE-cadherin. Genetic deletion of PLD2 impaired recovery from protease-activated receptor-1-activating peptide (PAR-1-AP)-induced lung vascular permeability and potentiated inflammation in vivo In human lung microvascular endothelial cells (HLMVECs), inhibition or deletion of PLD2, but not of PLD1, delayed endothelial barrier recovery after thrombin stimulation. Thrombin stimulation of HLMVECs increased co-localization of PLD2-generated PA and VE-cadherin at cell-cell adhesion junctions. Inhibition of PLD2 activity resulted in prolonged phosphorylation of Tyr-658 in VE-cadherin during the recovery phase 3 h post-thrombin challenge. Immunoprecipitation experiments revealed that after HLMVECs are thrombin stimulated, PLD2, VE-cadherin, and protein-tyrosine phosphatase nonreceptor type 14 (PTPN14), a PLD2-dependent protein-tyrosine phosphatase, strongly associate with each other. PTPN14 depletion delayed VE-cadherin dephosphorylation, reannealing of adherens junctions, and barrier function recovery. PLD2 inhibition attenuated PTPN14 activity and reversed PTPN14-dependent VE-cadherin dephosphorylation after thrombin stimulation. Our findings indicate that PLD2 promotes PTPN14-mediated dephosphorylation of VE-cadherin and that redistribution of VE-cadherin at adherens junctions is essential for recovery of endothelial barrier function after an edemagenic insult.
Asunto(s)
Antígenos CD/metabolismo , Barrera Alveolocapilar/metabolismo , Cadherinas/metabolismo , Células Endoteliales/metabolismo , Fosfolipasa D/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Uniones Adherentes/metabolismo , Animales , Barrera Alveolocapilar/citología , Células Endoteliales/citología , Femenino , Humanos , Masculino , Ratones , Fosforilación/efectos de los fármacos , Trombina/farmacologíaRESUMEN
Vascular endothelial (VE)-cadherin forms homotypic adherens junctions (AJs) in the endothelium, whereas N-cadherin forms heterotypic adhesion between endothelial cells and surrounding vascular smooth muscle cells and pericytes. Here we addressed the question whether both cadherin adhesion complexes communicate through intracellular signaling and contribute to the integrity of the endothelial barrier. We demonstrated that deletion of N-cadherin (Cdh2) in either endothelial cells or pericytes increases junctional endothelial permeability in lung and brain secondary to reduced accumulation of VE-cadherin at AJs. N-cadherin functions by increasing the rate of VE-cadherin recruitment to AJs and induces the assembly of VE-cadherin junctions. We identified the dual Rac1/RhoA Rho guanine nucleotide exchange factor (GEF) Trio as a critical component of the N-cadherin adhesion complex, which activates both Rac1 and RhoA signaling pathways at AJs. Trio GEF1-mediated Rac1 activation induces the recruitment of VE-cadherin to AJs, whereas Trio GEF2-mediated RhoA activation increases intracellular tension and reinforces Rac1 activation to promote assembly of VE-cadherin junctions and thereby establish the characteristic restrictive endothelial barrier.
Asunto(s)
Uniones Adherentes/metabolismo , Cadherinas/genética , Células Endoteliales/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Pericitos/metabolismo , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas/genética , Uniones Adherentes/ultraestructura , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Aorta/citología , Aorta/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Cadherinas/deficiencia , Cadherinas/metabolismo , Células Endoteliales/ultraestructura , Femenino , Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Pulmón/citología , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuropéptidos/genética , Neuropéptidos/metabolismo , Pericitos/ultraestructura , Permeabilidad , Fosfoproteínas/metabolismo , Cultivo Primario de Células , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoARESUMEN
Vascular endothelial cadherin (VE-cad) expression at endothelial adherens junctions (AJs) regulates vascular homeostasis. Here we show that endothelial A20 is required for VE-cad expression at AJs to maintain and repair the injured endothelial barrier. In endothelial cell (EC)-restricted Tnfaip3 (A20) knockout (A20∆EC ) mice, LPS challenge caused uncontrolled lung vascular leak and persistent sequestration of polymorphonuclear neutrophil (PMNs). Importantly, A20∆EC mice exhibited drastically reduced VE-cad expression in lungs compared with wild-type counterparts. Endothelial expression of wild-type A20 but not the deubiquitinase-inactive A20 mutant (A20C103A) prevented VE-cad ubiquitination, restored VE-cad expression, and suppressed lung vascular leak in A20∆EC mice. Interestingly, IRAK-M-mediated nuclear factor-κB (NF-κB) signaling downstream of TLR4 was required for A20 expression in ECs. interleukin-1 receptor-associated kinase M (IRAK-M) knockdown suppressed basal and LPS-induced A20 expression in ECs. Further, in vivo silencing of IRAK-M in mouse lung vascular ECs through the CRISPR-Cas9 system prevented expression of A20 and VE-cad while augmenting lung vascular leak. These results suggest that targeting of endothelial A20 is a potential therapeutic strategy to restore endothelial barrier integrity in the setting of acute lung injury.
RESUMEN
Acute lung injury is a leading cause of death in bacterial sepsis due to the wholesale destruction of the lung endothelial barrier, which results in protein-rich lung edema, influx of proinflammatory leukocytes, and intractable hypoxemia. Pyroptosis is a form of programmed lytic cell death that is triggered by inflammatory caspases, but little is known about its role in EC death and acute lung injury. Here, we show that systemic exposure to the bacterial endotoxin lipopolysaccharide (LPS) causes severe endothelial pyroptosis that is mediated by the inflammatory caspases, human caspases 4/5 in human ECs, or the murine homolog caspase-11 in mice in vivo. In caspase-11-deficient mice, BM transplantation with WT hematopoietic cells did not abrogate endotoxemia-induced acute lung injury, indicating a central role for nonhematopoietic caspase-11 in endotoxemia. Additionally, conditional deletion of caspase-11 in ECs reduced endotoxemia-induced lung edema, neutrophil accumulation, and death. These results establish the requisite role of endothelial pyroptosis in endotoxemic tissue injury and suggest that endothelial inflammatory caspases are an important therapeutic target for acute lung injury.
Asunto(s)
Caspasas/fisiología , Células Endoteliales/enzimología , Endotoxemia/enzimología , Lesión Pulmonar/enzimología , Piroptosis , Animales , Estudios de Casos y Controles , Caspasas Iniciadoras , Células Cultivadas , Endotelio Vascular/patología , Endotoxemia/inmunología , Femenino , Humanos , Interleucina-1beta/metabolismo , Lipopolisacáridos/farmacología , Pulmón/enzimología , Pulmón/inmunología , Pulmón/patología , Lesión Pulmonar/inmunología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor Toll-Like 4/metabolismoRESUMEN
Vascular endothelial protein tyrosine phosphatase (VE-PTP) stabilizes endothelial adherens junctions (AJs) through constitutive dephosphorylation of VE-cadherin. Here we investigated the role of stromal interaction molecule 1 (STIM1) activation of store-operated Ca2+ entry (SOCE) in regulating AJ assembly. We observed that SOCE induced by STIM1 activated Pyk2 in human lung microvascular endothelial cells (ECs) and induced tyrosine phosphorylation of VE-PTP at Y1981. Pyk2-induced tyrosine phosphorylation of VE-PTP promoted Src binding to VE-PTP, Src activation, and subsequent VE-cadherin phosphorylation and thereby increased the endothelial permeability response. The increase in permeability was secondary to disassembly of AJs. Pyk2-mediated responses were blocked in EC-restricted Stim1 knockout mice, indicating the requirement for STIM1 in initiating the signaling cascade. A peptide derived from the Pyk2 phosphorylation site on VE-PTP abolished the STIM1/SOCE-activated permeability response. Thus Pyk2 activation secondary to STIM1-induced SOCE causes tyrosine phosphorylation of VE-PTP, and VE-PTP, in turn, binds to and activates Src, thereby phosphorylating VE-cadherin to increase endothelial permeability through disassembly of AJs. Our results thus identify a novel signaling mechanism by which STIM1-induced Ca2+ signaling activates Pyk2 to inhibit the interaction of VE-PTP and VE-cadherin and hence increase endothelial permeability. Therefore, targeting the Pyk2 activation pathway may be a potentially important anti-inflammatory strategy.
Asunto(s)
Uniones Adherentes/metabolismo , Calcio/metabolismo , Quinasa 2 de Adhesión Focal/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Permeabilidad Capilar , Permeabilidad de la Membrana Celular , Células Endoteliales/metabolismo , Activación Enzimática , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Humanos , Ratones Endogámicos C57BL , Microvasos/citología , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Péptidos/metabolismo , Fosforilación , Fosfotirosina/metabolismo , Receptor PAR-1/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
The NF-κB pathway is central to the regulation of inflammation. Here, we demonstrate that the low-output nitric oxide (NO) synthase 1 (NOS1 or nNOS) plays a critical role in the inflammatory response by promoting the activity of NF-κB. Specifically, NOS1-derived NO production in macrophages leads to proteolysis of suppressor of cytokine signaling 1 (SOCS1), alleviating its repression of NF-κB transcriptional activity. As a result, NOS1(-/-) mice demonstrate reduced cytokine production, lung injury, and mortality when subjected to two different models of sepsis. Isolated NOS1(-/-) macrophages demonstrate similar defects in proinflammatory transcription on challenge with Gram-negative bacterial LPS. Consistently, we found that activated NOS1(-/-) macrophages contain increased SOCS1 protein and decreased levels of p65 protein compared with wild-type cells. NOS1-dependent S-nitrosation of SOCS1 impairs its binding to p65 and targets SOCS1 for proteolysis. Treatment of NOS1(-/-) cells with exogenous NO rescues both SOCS1 degradation and stabilization of p65 protein. Point mutation analysis demonstrated that both Cys147 and Cys179 on SOCS1 are required for its NO-dependent degradation. These findings demonstrate a fundamental role for NOS1-derived NO in regulating TLR4-mediated inflammatory gene transcription, as well as the intensity and duration of the resulting host immune response.
Asunto(s)
FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Óxido Nítrico/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Animales , Citocinas/metabolismo , Humanos , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , FN-kappa B/genética , Óxido Nítrico Sintasa de Tipo I/genética , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Sepsis/genética , Sepsis/mortalidad , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genética , Receptor Toll-Like 4/metabolismoRESUMEN
The mechanisms by which the microtubule cytoskeleton regulates the permeability of endothelial barrier are not well understood. Here, we demonstrate that microtubule-associated end-binding protein 3 (EB3), a core component of the microtubule plus-end protein complex, binds to inositol 1,4,5-trisphosphate receptors (IP3Rs) through an S/TxIP EB-binding motif. In endothelial cells, α-thrombin, a pro-inflammatory mediator that stimulates phospholipase Cß, increases the cytosolic Ca(2+) concentration and elicits clustering of IP3R3s. These responses, and the resulting Ca(2+)-dependent phosphorylation of myosin light chain, are prevented by depletion of either EB3 or mutation of the TxIP motif of IP3R3 responsible for mediating its binding to EB3. We also show that selective EB3 gene deletion in endothelial cells of mice abrogates α-thrombin-induced increase in endothelial permeability. We conclude that the EB3-mediated interaction of IP3Rs with microtubules controls the assembly of IP3Rs into effective Ca(2+) signaling clusters, which thereby regulate microtubule-dependent endothelial permeability.
Asunto(s)
Señalización del Calcio , Células Endoteliales/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Animales , Células CHO , Permeabilidad Capilar , Cricetinae , Cricetulus , Células HEK293 , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/genética , Unión ProteicaRESUMEN
The integrity of the lung alveolar epithelial barrier is required for the gas exchange and is important for immune regulation. Alveolar epithelial barrier is composed of flat type I cells, which make up approximately 95% of the gas-exchange surface, and cuboidal type II cells, which secrete surfactants and modulate lung immunity. p120-catenin (p120; gene symbol CTNND1) is an important component of adherens junctions of epithelial cells; however, its function in lung alveolar epithelial barrier has not been addressed in genetic models. Here, we created an inducible type II cell-specific p120-knockout mouse (p120EKO). The mutant lungs showed chronic inflammation, and the alveolar epithelial barrier was leaky to (125)I-albumin tracer compared to wild type. The mutant lungs also demonstrated marked infiltration of inflammatory cells and activation of NF-κB. Intracellular adhesion molecule 1, Toll-like receptor 4, and macrophage inflammatory protein 2 were all up-regulated. p120EKO lungs showed increased expression of the surfactant proteins Sp-B, Sp-C, and Sp-D, and displayed severe inflammation after pneumonia caused by Pseudomonas aeruginosa compared with wild type. In p120-deficient type II cell monolayers, we observed reduced transepithelial resistance compared to control, consistent with formation of defective adherens junctions. Thus, although type II cells constitute only 5% of the alveolar surface area, p120 expressed in these cells plays a critical role in regulating the innate immunity of the entire lung.
Asunto(s)
Células Epiteliales Alveolares/inmunología , Cateninas/inmunología , Inmunidad Innata/inmunología , Pulmón/inmunología , Células Epiteliales Alveolares/metabolismo , Animales , Western Blotting , Permeabilidad Capilar/inmunología , Cateninas/metabolismo , Femenino , Inmunohistoquímica , Inflamación/inmunología , Inflamación/metabolismo , Pulmón/metabolismo , Masculino , Ratones , Ratones Noqueados , Alveolos Pulmonares/inmunología , Alveolos Pulmonares/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Catenina deltaRESUMEN
Vascular endothelial barrier dysfunction underlies diseases such as acute respiratory distress syndrome (ARDS), characterized by edema and inflammatory cell infiltration. The transcription factor HIF2α is highly expressed in vascular endothelial cells (ECs) and may regulate endothelial barrier function. Here, we analyzed promoter sequences of genes encoding proteins that regulate adherens junction (AJ) integrity and determined that vascular endothelial protein tyrosine phosphatase (VE-PTP) is a HIF2α target. HIF2α-induced VE-PTP expression enhanced dephosphorylation of VE-cadherin, which reduced VE-cadherin endocytosis and thereby augmented AJ integrity and endothelial barrier function. Mice harboring an EC-specific deletion of Hif2a exhibited decreased VE-PTP expression and increased VE-cadherin phosphorylation, resulting in defective AJs. Mice lacking HIF2α in ECs had increased lung vascular permeability and water content, both of which were further exacerbated by endotoxin-mediated injury. Treatment of these mice with Fg4497, a prolyl hydroxylase domain 2 (PHD2) inhibitor, activated HIF2α-mediated transcription in a hypoxia-independent manner. HIF2α activation increased VE-PTP expression, decreased VE-cadherin phosphorylation, promoted AJ integrity, and prevented the loss of endothelial barrier function. These findings demonstrate that HIF2α enhances endothelial barrier integrity, in part through VE-PTP expression and the resultant VE-cadherin dephosphorylation-mediated assembly of AJs. Moreover, activation of HIF2α/VE-PTP signaling via PHD2 inhibition has the potential to prevent the formation of leaky vessels and edema in inflammatory diseases such as ARDS.
Asunto(s)
Lesión Pulmonar Aguda/metabolismo , Uniones Adherentes/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células Endoteliales/metabolismo , Síndrome de Dificultad Respiratoria/metabolismo , Transducción de Señal , Lesión Pulmonar Aguda/patología , Uniones Adherentes/genética , Uniones Adherentes/patología , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular , Células Endoteliales/patología , Regulación Enzimológica de la Expresión Génica/genética , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia/antagonistas & inhibidores , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Ratones , Ratones Noqueados , Fosforilación/genética , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/biosíntesis , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/genética , Síndrome de Dificultad Respiratoria/genética , Síndrome de Dificultad Respiratoria/patologíaRESUMEN
Stromal interacting molecule 1 (STIM1) regulates store-operated Ca(2+) entry (SOCE). Here we show that STIM1 expression in endothelial cells (ECs) is increased during sepsis and, therefore, contributes to hyperpermeability. LPS induced STIM1 mRNA and protein expression in human and mouse lung ECs. The induced STIM1 expression was associated with augmented SOCE as well as a permeability increase in both in vitro and in vivo models. Because activation of both the NF-κB and p38 MAPK signaling pathways downstream of TLR4 amplifies vascular inflammation, we studied the influence of these two pathways on LPS-induced STIM1 expression. Inhibition of either NF-κB or p38 MAPK activation by pharmacological agents prevented LPS-induced STIM1 expression. Silencing of the NF-κB proteins (p65/RelA or p50/NF-κB1) or the p38 MAPK isoform p38α prevented LPS-induced STIM1 expression and increased SOCE in ECs. In support of these findings, we found NF-κB and AP1 binding sites in the 5'-regulatory region of human and mouse STIM1 genes. Further, we demonstrated that LPS induced time-dependent binding of the transcription factors NF-κB (p65/RelA) and AP1 (c-Fos/c-Jun) to the STIM1 promoter. Interestingly, silencing of c-Fos, but not c-Jun, markedly reduced LPS-induced STIM1 expression in ECs. We also observed that silencing of p38α prevented c-Fos expression in response to LPS in ECs, suggesting that p38α signaling mediates the expression of c-Fos. These results support the proposal that cooperative signaling of both NF-κB and AP1 (via p38α) amplifies STIM1 expression in ECs and, thereby, contributes to the lung vascular hyperpermeability response during sepsis.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Endotoxinas/farmacología , Proteínas de la Membrana/metabolismo , FN-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Transducción de Señal , Factor de Transcripción AP-1/metabolismo , Animales , Secuencia de Bases , Permeabilidad Capilar , Células Cultivadas , Cartilla de ADN , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Molécula de Interacción Estromal 1 , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Impairment of tissue fluid homeostasis and migration of inflammatory cells across the vascular endothelial barrier are crucial factors in the pathogenesis of acute lung injury (ALI). The goal for treatment of ALI is to target pathways that lead to profound dysregulation of the lung endothelial barrier. Although studies have shown that chemical epigenetic modifiers can limit lung inflammation in experimental ALI models, studies to date have not examined efficacy of a combination of DNA methyl transferase inhibitor 5-Aza 2-deoxycytidine and histone deacetylase inhibitor trichostatin A (herein referred to as Aza+TSA) after endotoxemia-induced mouse lung injury. We tested the hypothesis that treatment with Aza+TSA after lipopolysaccharide induction of ALI through epigenetic modification of lung endothelial cells prevents inflammatory lung injury. Combinatorial treatment with Aza+TSA mitigated the increased endothelial permeability response after lipopolysaccharide challenge. In addition, we observed reduced lung inflammation and lung injury. Aza+TSA also significantly reduced mortality in the ALI model. The protection was ascribed to inhibition of the eNOS-Cav1-MLC2 signaling pathway and enhanced acetylation of histone markers on the vascular endothelial-cadherin promoter. In summary, these data show for the first time the efficacy of combinatorial Aza+TSA therapy in preventing ALI in lipopolysaccharide-induced endotoxemia and raise the possibility of an essential role of DNA methyl transferase and histone deacetylase in the mechanism of ALI.
Asunto(s)
Lesión Pulmonar Aguda/patología , Azacitidina/análogos & derivados , Permeabilidad Capilar/efectos de los fármacos , Inhibidores Enzimáticos/administración & dosificación , Ácidos Hidroxámicos/administración & dosificación , Pulmón/efectos de los fármacos , Acetilación , Lesión Pulmonar Aguda/enzimología , Animales , Azacitidina/administración & dosificación , Western Blotting , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Inmunoprecipitación de Cromatina , Decitabina , Modelos Animales de Enfermedad , Quimioterapia Combinada , Células Endoteliales/efectos de los fármacos , Endotoxemia/enzimología , Endotoxemia/patología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Etiquetado Corte-Fin in Situ , Inflamación/enzimología , Inflamación/patología , Masculino , Metilación , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The heterotrimeric G protein Gα13 transduces signals from G protein-coupled receptors (GPCRs) to induce cell spreading, differentiation, migration, and cell polarity. Here, we describe a novel GPCR-independent function of Gα13 in regulating the stability of endothelial cell adherens junctions (AJs). We observed that the oxidant H2O2, which is released in response to multiple proinflammatory mediators, induced the interaction of Gα13 with VE-cadherin. Gα13 binding to VE-cadherin in turn induced Src activation and VE-cadherin phosphorylation at Tyr 658, the p120-catenin binding site thought to be responsible for VE-cadherin internalization. Inhibition of Gα13-VE-cadherin interaction using an interfering peptide derived from the Gα13 binding motif on VE-cadherin abrogated the disruption of AJs in response to inflammatory mediators. These studies identify a unique role of Gα13 binding to VE-cadherin in mediating VE-cadherin internalization and endothelial barrier disruption and inflammation.
Asunto(s)
Uniones Adherentes/metabolismo , Antígenos CD/metabolismo , Cadherinas/metabolismo , Células Endoteliales/metabolismo , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Inflamación/metabolismo , Animales , Biotinilación , Western Blotting , Impedancia Eléctrica , Endocitosis/fisiología , Células Endoteliales/citología , Azul de Evans , Subunidades alfa de la Proteína de Unión al GTP G12-G13/deficiencia , Genes src/genética , Peróxido de Hidrógeno/metabolismo , Inmunoprecipitación , Ratones , Ratones Noqueados , Permeabilidad , Fosforilación , Interferencia de ARNRESUMEN
Adult stem cell-based therapy is a promising novel approach for treatment of acute lung injury. Here we investigated the therapeutic potential of freshly isolated human umbilical cord blood CD34(+) progenitor cells (fCB-CD34(+) cells) in a mouse model of acute lung injury. At 3 h post-lipopolysaccharide (LPS) challenge, fCB-CD34(+) cells were transplanted i.v. to mice while CD34(-) cells or PBS were administered as controls in separate cohorts of mice. We observed that fCB-CD34(+) cell treatment inhibited lung vascular injury evident by decreased lung vascular permeability. In contrast, CD34(-) cells had no effects on lung vascular injury. Lung inflammation determined by myeloperoxidase activity, neutrophil sequestration and expression of pro-inflammatory mediators was attenuated in fCB-CD34(+) cell-treated mice at 26 h post-LPS challenge compared to PBS or CD34(-) cell-treated controls. Importantly, lung inflammation in fCB-CD34(+) cell-treated mice was returned to normal levels as seen in basal mice at 52 h post-LPS challenge whereas PBS or CD34(-) cell-treated control mice exhibited persistent lung inflammation. Accordingly, fCB-CD34(+) cell-treated mice exhibited a marked increase of survival rate. Employing in vivo 5-bromo-2'-deoxyuridine incorporation assay, we found a drastic induction of lung endothelial proliferation in fCB-CD34(+) cell-treated mice at 52 h post-LPS compared to PBS or CD34(-) cell-treated controls, which contributed to restoration of vascular integrity and thereby inhibition of lung inflammation. Taken together, these data have demonstrated the protective effects of fCB-CD34(+) cell on acute lung injury induced by LPS challenge, suggesting fCB-CD34(+) cells are an important source of stem cells for the treatment of acute lung injury.
Asunto(s)
Antígenos CD34/metabolismo , Sangre Fetal/citología , Lipopolisacáridos/efectos adversos , Lesión Pulmonar/inducido químicamente , Lesión Pulmonar/terapia , Trasplante de Células Madre , Células Madre/citología , Animales , Proliferación Celular , Separación Celular , Células Endoteliales/citología , Humanos , Inflamación/inducido químicamente , Inflamación/patología , Inflamación/terapia , Pulmón/irrigación sanguínea , Pulmón/efectos de los fármacos , Pulmón/patología , Lesión Pulmonar/patología , Ratones , Análisis de SupervivenciaRESUMEN
Here we found that the transcription repressor DREAM bound to the promoter of the gene encoding A20 to repress expression of this deubiquitinase that suppresses inflammatory NF-κB signaling. DREAM-deficient mice displayed persistent and unchecked A20 expression in response to endotoxin. DREAM functioned by transcriptionally repressing A20 through binding to downstream regulatory elements (DREs). In contrast, binding of the transcription factor USF1 to the DRE-associated E-box domain in the gene encoding A20 activated its expression in response to inflammatory stimuli. Our studies define the critical opposing functions of DREAM and USF1 in inhibiting and inducing A20 expression, respectively, and thereby the strength of NF-κB signaling. Targeting of DREAM to induce USF1-mediated A20 expression is therefore a potential anti-inflammatory strategy for the treatment of diseases associated with unconstrained NF-κB activity, such as acute lung injury.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Proteínas de Interacción con los Canales Kv/metabolismo , Proteínas Represoras/metabolismo , Ubiquitina-Proteína Ligasas/biosíntesis , Factores Estimuladores hacia 5'/metabolismo , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/metabolismo , Animales , Inmunoprecipitación de Cromatina , Cisteína Endopeptidasas , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/inmunología , Immunoblotting , Inflamación/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Excessive reactive oxygen/nitrogen species have been associated with the onset, progression, and outcome of sepsis, both in preclinical and clinical studies. However, the signaling pathways regulating oxidative/nitrative stress in the pathogenesis of sepsis-induced acute lung injury and acute respiratory distress syndrome are not fully understood. Employing the novel mouse model with genetic deletions of both caveolin-1 (Cav1) and adiponectin (ADPN) [double knockout (DKO) mice], we have demonstrated the critical role of Cav1 and ADPN signaling cross talk in regulating oxidative/nitrative stress and resulting inflammatory lung injury following LPS challenge. In contrast to the inhibited inflammatory lung injury in Cav1(-/-) mice, we observed severe lung inflammation and markedly increased lung vascular permeability in DKO mice in response to LPS challenge. Accordingly, the DKO mice exhibited an 80% mortality rate following a sublethal dose of LPS challenge. At basal state, loss of Cav1 and ADPN resulted in a drastic increase of oxidative stress and resultant nitrative stress in DKO lungs. Scavenging of superoxide by pretreating the DKO mice with MnTMPYP (a superoxide dismutase mimetic) restored the inflammatory responses to LPS challenge including reduced lung myeloperoxidase activity and vascular permeability. Thus oxidative/nitrative stress collectively modulated by Cav1 and ADPN signalings is a critical determinant of inflammatory lung injury in response to LPS challenge.
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Lesión Pulmonar Aguda/inmunología , Adiponectina/metabolismo , Caveolina 1/metabolismo , Estrés Oxidativo/inmunología , Neumonía/inmunología , Lesión Pulmonar Aguda/genética , Adiponectina/deficiencia , Adiponectina/genética , Adiponectina/inmunología , Animales , Permeabilidad Capilar/genética , Permeabilidad Capilar/inmunología , Caveolina 1/deficiencia , Caveolina 1/genética , Lipopolisacáridos , Pulmón/inmunología , Pulmón/patología , Errores Innatos del Metabolismo/inmunología , Ratones , Ratones Noqueados , Oxidación-Reducción , Neumonía/genética , Especies de Nitrógeno Reactivo/inmunología , Especies Reactivas de Oxígeno/inmunología , Sepsis/genética , Sepsis/inmunología , Transducción de Señal/inmunologíaRESUMEN
RATIONALE: Oxidants generated by activated endothelial cells are known to induce apoptosis, a pathogenic feature of vascular injury and inflammation from multiple pathogeneses. The melastatin-family transient receptor potential 2 (TRPM2) channel is an oxidant-sensitive Ca2+ permeable channel implicated in mediating apoptosis; however, the mechanisms of gating of the supranormal Ca2+ influx required for initiating of apoptosis are not understood. OBJECTIVE: Here, we addressed the role of TRPM2 and its interaction with the short splice variant TRPM2 short variant (TRPM2-S) in mediating the Ca2+ entry burst required for induction of endothelial cell apoptosis. METHODS AND RESULTS: We observed that TRPM2-S was basally associated with TRPM2 in the endothelial plasmalemma, and this interaction functioned to suppress TRPM2-dependent Ca2+ gating constitutively. Reactive oxygen species production in endothelial cells or directly applying reactive oxygen species induced protein kinase C-α activation and phosphorylation of TRPM2 at Ser 39. This in turn stimulated a large entry of Ca2+ and activated the apoptosis pathway. A similar TRPM2-dependent endothelial apoptosis mechanism was seen in intact vessels. The protein kinase C-α-activated phosphoswitch opened the TRPM2 channel to allow large Ca2+ influx by releasing TRPM2-S inhibition of TRPM2, which in turn activated caspase-3 and cleaved the caspase substrate poly(ADP-ribose) polymerase. CONCLUSIONS: Here, we describe a fundamental mechanism by which activation of the trp superfamily TRPM2 channel induces apoptosis of endothelial cells. The signaling mechanism involves reactive oxygen species-induced protein kinase C-α activation resulting in phosphorylation of TRPM2-S that allows enhanced TRPM2-mediated gating of Ca2+ and activation of the apoptosis program. Strategies aimed at preventing the uncoupling of TRPM2-S from TRPM2 and subsequent Ca2+ gating during oxidative stress may mitigate endothelial apoptosis and its consequences in mediating vascular injury and inflammation.
Asunto(s)
Apoptosis/genética , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Isoformas de Proteínas/metabolismo , Empalme del ARN/genética , Canales Catiónicos TRPM/metabolismo , Animales , Señalización del Calcio/genética , Células Cultivadas , Variación Genética , Humanos , Ratones , Ratones Noqueados , Familia de Multigenes , Unión Proteica/genética , Isoformas de Proteínas/genética , Canales Catiónicos TRPM/genéticaRESUMEN
von Willebrand factor (vWF) secretion by endothelial cells (ECs) is essential for hemostasis and thrombosis; however, the molecular mechanisms are poorly understood. Interestingly, we observed increased bleeding in EC-Gα13(-/-);Gα12(-/-) mice that could be normalized by infusion of human vWF. Blood from Gα12(-/-) mice exhibited significantly reduced vWF levels but normal vWF multimers and impaired laser-induced thrombus formation, indicating that Gα12 plays a prominent role in EC vWF secretion required for hemostasis and thrombosis. In isolated buffer-perfused mouse lungs, basal vWF levels were significantly reduced in Gα12(-/-), whereas thrombin-induced vWF secretion was defective in both EC-Gαq(-/-);Gα11(-/-) and Gα12(-/-) mice. Using siRNA in cultured human umbilical vein ECs and human pulmonary artery ECs, depletion of Gα12 and soluble N-ethylmaleimide-sensitive-fusion factor attachment protein α (α-SNAP), but not Gα13, inhibited both basal and thrombin-induced vWF secretion, whereas overexpression of activated Gα12 promoted vWF secretion. In Gαq, p115 RhoGEF, and RhoA-depleted human umbilical vein ECs, thrombin-induced vWF secretion was reduced by 40%, whereas basal secretion was unchanged. Finally, in vitro binding assays revealed that Gα12 N-terminal residues 10-15 mediated the binding of Gα12 to α-SNAP, and an engineered α-SNAP binding-domain minigene peptide blocked basal and evoked vWF secretion. Discovery of obligatory and complementary roles of Gα12 and Gαq/11 in basal vs evoked EC vWF secretion may provide promising new therapeutic strategies for treatment of thrombotic disease.