RESUMEN
The regulation of polymorphonuclear leukocyte (PMN) function by mechanical forces encountered during their migration across restrictive endothelial cell junctions is not well understood. Using genetic, imaging, microfluidic, and in vivo approaches, we demonstrated that the mechanosensor Piezo1 in PMN plasmalemma induced spike-like Ca2+ signals during trans-endothelial migration. Mechanosensing increased the bactericidal function of PMN entering tissue. Mice in which Piezo1 in PMNs was genetically deleted were defective in clearing bacteria, and their lungs were predisposed to severe infection. Adoptive transfer of Piezo1-activated PMNs into the lungs of Pseudomonas aeruginosa-infected mice or exposing PMNs to defined mechanical forces in microfluidic systems improved bacterial clearance phenotype of PMNs. Piezo1 transduced the mechanical signals activated during transmigration to upregulate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4, crucial for the increased PMN bactericidal activity. Thus, Piezo1 mechanosensing of increased PMN tension, while traversing the narrow endothelial adherens junctions, is a central mechanism activating the host-defense function of transmigrating PMNs.
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Movimiento Celular , Pulmón , Mecanotransducción Celular , Neutrófilos , Animales , Ratones , Membrana Celular , Canales Iónicos/genética , Neutrófilos/metabolismo , Neutrófilos/microbiología , Actividad Bactericida de la Sangre/genética , Mecanotransducción Celular/genéticaRESUMEN
Airway epithelial cells (AECs) secrete innate immune cytokines that regulate adaptive immune effector cells. In allergen-sensitized humans and mice, the airway and alveolar microenvironment is enriched with colony stimulating factor-1 (CSF1) in response to allergen exposure. In this study we found that AEC-derived CSF1 had a critical role in the production of allergen reactive-IgE production. Furthermore, spatiotemporally secreted CSF1 regulated the recruitment of alveolar dendritic cells (DCs) and enhanced the migration of conventional DC2s (cDC2s) to the draining lymph node in an interferon regulatory factor 4 (IRF4)-dependent manner. CSF1 selectively upregulated the expression of the chemokine receptor CCR7 on the CSF1R+ cDC2, but not the cDC1, population in response to allergen stimuli. Our data describe the functional specification of CSF1-dependent DC subsets that link the innate and adaptive immune responses in T helper 2 (Th2) cell-mediated allergic lung inflammation.
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Alérgenos/inmunología , Células Dendríticas/inmunología , Factor Estimulante de Colonias de Macrófagos/inmunología , Receptores CCR7/biosíntesis , Mucosa Respiratoria/citología , Mucosa Respiratoria/inmunología , Animales , Línea Celular , Movimiento Celular/inmunología , Células Dendríticas/clasificación , Células Epiteliales/citología , Células Epiteliales/inmunología , Humanos , Inmunidad Innata/inmunología , Inmunoglobulina E/inmunología , Factores Reguladores del Interferón/inmunología , Ganglios Linfáticos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células RAW 264.7 , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Células Th2/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
Sphingosine kinase 1 (SPHK1) has been shown to play a key role in the pathogenesis of asthma where SPHK1-generated sphingosine-1-phosphate (S1P) is known to mediate innate and adaptive immunity while promoting mast cell degranulation. Goblet cell metaplasia (GCM) contributes to airway obstruction in asthma and has been demonstrated in animal models. We investigated the role of PF543, a SPHK1-specific inhibitor, in preventing the pathogenesis of GCM using a murine (C57BL/6) model of allergen-induced acute asthma. Treatment with PF543 before triple allergen exposure (DRA: House dust mite, Ragweed pollen, and Aspergillus) reduced inflammation, eosinophilic response, and GCM followed by reduced airway hyperreactivity to intravenous methacholine. Furthermore, DRA exposure was associated with increased expression of SPHK1 in the airway epithelium which was reduced by PF543. DRA-induced reduction of acetylated α-tubulin in airway epithelium was associated with an increased expression of NOTCH2 and SPDEF which was prevented by PF543. In vitro studies using human primary airway epithelial cells showed that inhibition of SPHK1 using PF543 prevented an allergen-induced increase of both NOTCH2 and SPDEF. siRNA silencing of SPHK1 prevented the allergen-induced increase of both NOTCH2 and SPDEF. NOTCH2 silencing was associated with a reduction of SPDEF but not that of SPHK1 upon allergen exposure. Our studies demonstrate that inhibition of SPHK1 protected allergen-challenged airways by preventing GCM and airway hyperreactivity, associated with downregulation of the NOTCH2-SPDEF signaling pathway. This suggests a potential novel link between SPHK1, GCM, and airway remodeling in asthma.NEW & NOTEWORTHY The role of SPHK1-specific inhibitor, PF543, in preventing goblet cell metaplasia (GCM) and airway hyperreactivity (AHR) is established in an allergen-induced mouse model. This protection was associated with the downregulation of NOTCH2-SPDEF signaling pathway, suggesting a novel link between SPHK1, GCM, and AHR.
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Asma , Células Caliciformes , Lisofosfolípidos , Fosfotransferasas (Aceptor de Grupo Alcohol) , Pirrolidinas , Esfingosina/análogos & derivados , Sulfonas , Animales , Humanos , Ratones , Células Caliciformes/metabolismo , Ratones Endogámicos C57BL , Asma/patología , Epitelio/metabolismo , Factores de Transcripción/metabolismo , Metaplasia/metabolismo , Metaplasia/patología , Alérgenos , MetanolRESUMEN
Chronic cigarette smoke exposure decreases lung expression of WWOX which is known to protect the endothelial barrier during infectious models of acute respiratory distress syndrome (ARDS). Proteomic analysis of WWOX-silenced endothelial cells (ECs) was done using tandem mass tag mass spectrometry (TMT-MS). WWOX-silenced ECs as well as those isolated from endothelial cell Wwox knockout (EC Wwox KO) mice were subjected to cyclic stretch (18% elongation, 0.5 Hz, 4 h). Cellular lysates and media supernatant were harvested for assays of cellular signaling, protein expression, and cytokine release. These were repeated with dual silencing of WWOX and zyxin. Control and EC Wwox KO mice were subjected to high tidal volume ventilation. Bronchoalveolar lavage fluid and mouse lung tissue were harvested for cellular signaling, cytokine secretion, and histological assays. TMT-MS revealed upregulation of zyxin expression during WWOX knockdown which predicted a heightened inflammatory response to mechanical stretch. WWOX-silenced ECs and ECs isolated from EC Wwox mice displayed significantly increased cyclic stretch-mediated secretion of various cytokines (IL-6, KC/IL-8, IL-1ß, and MCP-1) relative to controls. This was associated with increased ERK and JNK phosphorylation but decreased p38 mitogen-activated kinases (MAPK) phosphorylation. EC Wwox KO mice subjected to VILI sustained a greater degree of injury than corresponding controls. Silencing of zyxin during WWOX knockdown abrogated stretch-induced increases in IL-8 secretion but not in IL-6. Loss of WWOX function in ECs is associated with a heightened inflammatory response during mechanical stretch that is associated with increased MAPK phosphorylation and appears, in part, to be dependent on the upregulation of zyxin.NEW & NOTEWORTHY Prior tobacco smoke exposure is associated with an increased risk of acute respiratory distress syndrome (ARDS) during critical illness. Our laboratory is investigating one of the gene expression changes that occurs in the lung following smoke exposure: WWOX downregulation. Here we describe changes in protein expression associated with WWOX knockdown and its influence on ventilator-induced ARDS in a mouse model.
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Células Endoteliales , Inflamación , Ratones Noqueados , Lesión Pulmonar Inducida por Ventilación Mecánica , Oxidorreductasa que Contiene Dominios WW , Animales , Oxidorreductasa que Contiene Dominios WW/metabolismo , Oxidorreductasa que Contiene Dominios WW/genética , Ratones , Células Endoteliales/metabolismo , Células Endoteliales/patología , Inflamación/metabolismo , Inflamación/patología , Lesión Pulmonar Inducida por Ventilación Mecánica/metabolismo , Lesión Pulmonar Inducida por Ventilación Mecánica/patología , Lesión Pulmonar Inducida por Ventilación Mecánica/genética , Citocinas/metabolismo , Ratones Endogámicos C57BL , Técnicas de Silenciamiento del Gen , Masculino , Pulmón/metabolismo , Pulmón/patología , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
In patients with sickle cell disease (SCD), acute chest syndrome (ACS) is a common form of acute lung injury and a major cause of morbidity and mortality. The pathophysiology of ACS is complex, and hemin, the prosthetic moiety of hemoglobin, has been implicated in endothelial cell (EC) activation and subsequent acute lung injury (ALI) and ACS in vitro and in animal studies. Here, we examined the role of cortactin (CTTN), a cytoskeletal protein that regulates EC function, in response to hemin-induced ALI and ACS. Cortactin heterozygous (Cttn+/-) mice (n = 8) and their wild-type siblings (n = 8) were irradiated and subsequently received bone marrow cells (BMCs) extruded from the femurs of SCD mice (SS) to generate SS Cttn+/- and SS CttnWT chimeras. Following hemoglobin electrophoretic proof of BMC transplantation, the mice received 35 µmol/kg of hemin. Within 24 h, surviving mice were euthanized, and bronchoalveolar fluid (BAL) and lung samples were analyzed. For in vitro studies, human lung microvascular endothelial cells (HLMVECs) were used to determine hemin-induced changes in gene expression and reactive oxygen species (ROS) generation in cortactin deficiency and control conditions. When compared with wild-type littermates, the mortality for SS Cttn+/- mice trended to be lower after hemin infusion and these mice exhibited less severe lung injury and less necroptotic cell death. In vitro studies confirmed that cortactin deficiency is protective against hemin-induced injury in HMLVECs, by decreasing protein expression of p38/HSP27, improving cell barrier function, and decreasing the production of ROS. Further studies examining the role of CTTN in ACS are warranted and may open a new avenue of potential treatment for this devastating disease.
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Lesión Pulmonar Aguda , Anemia de Células Falciformes , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/prevención & control , Anemia de Células Falciformes/complicaciones , Animales , Cortactina/genética , Cortactina/metabolismo , Células Endoteliales/metabolismo , Hemina/metabolismo , Hemina/farmacología , Humanos , Ratones , Especies Reactivas de Oxígeno/metabolismoRESUMEN
INTRODUCTION: Neonatal lung injury as a consequence of hyperoxia (HO) therapy and ventilator care contribute to the development of bronchopulmonary dysplasia (BPD). Increased expression and activity of lysyl oxidase (LOX), a key enzyme that cross-links collagen, was associated with increased sphingosine kinase 1 (SPHK1) in human BPD. We, therefore, examined closely the link between LOX and SPHK1 in BPD. METHOD: The enzyme expression of SPHK1 and LOX were assessed in lung tissues of human BPD using immunohistochemistry and quantified (Halo). In vivo studies were based on Sphk1-/- and matched wild type (WT) neonatal mice exposed to HO while treated with PF543, an inhibitor of SPHK1. In vitro mechanistic studies used human lung microvascular endothelial cells (HLMVECs). RESULTS: Both SPHK1 and LOX expressions were increased in lungs of patients with BPD. Tracheal aspirates from patients with BPD had increased LOX, correlating with sphingosine-1-phosphate (S1P) levels. HO-induced increase of LOX in lungs were attenuated in both Sphk1-/- and PF543-treated WT mice, accompanied by reduced collagen staining (sirius red). PF543 reduced LOX activity in both bronchoalveolar lavage fluid and supernatant of HLMVECs following HO. In silico analysis revealed STAT3 as a potential transcriptional regulator of LOX. In HLMVECs, following HO, ChIP assay confirmed increased STAT3 binding to LOX promoter. SPHK1 inhibition reduced phosphorylation of STAT3. Antibody to S1P and siRNA against SPNS2, S1P receptor 1 (S1P1) and STAT3 reduced LOX expression. CONCLUSION: HO-induced SPHK1/S1P signalling axis plays a critical role in transcriptional regulation of LOX expression via SPNS2, S1P1 and STAT3 in lung endothelium.
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Hiperoxia , Lesión Pulmonar , Animales , Células Endoteliales , Humanos , Ratones , Fosfotransferasas (Aceptor de Grupo Alcohol) , Proteína-Lisina 6-Oxidasa , Factor de Transcripción STAT3RESUMEN
Retinoblastoma (RB1) protein is a multifunctional protein that plays an important role in cell cycle regulation and cell differentiation, including adipogenesis. A detailed literature search to understand the role of RB1 in adipogenesis revealed that the nature of the RB1 inactivation (in vivo/in vitro) led to differences in adipogenesis. The majority of these studies were animal-based, and the only study in humans employed an in vitro mode of RB1 inactivation. To overcome these differences and lack of human studies, we sought to explore the role of RB1 in adipogenesis using orbital adipose mesenchymal stem cells (OAMSCs) from patients with retinoblastoma that innately carry a heterozygous RB1 mutation. We hypothesized that these patient-derived RB1 mutant OAMSCs can model in vivo RB1 inactivation in humans. Our study revealed increased adipogenesis with a bias toward brown adipogenesis in the RB1 mutant in addition to an increased number of adipocytes in the mitotic phase.
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Adipogénesis , Proteínas de Unión a Retinoblastoma , Retinoblastoma , Células Madre , Ubiquitina-Proteína Ligasas , Adipocitos , Adipogénesis/genética , Diferenciación Celular , Humanos , Retinoblastoma/genética , Proteínas de Unión a Retinoblastoma/genética , Células Madre/citología , Células Madre/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Premature infants are born with developing lungs burdened by surfactant deficiency and a dearth of antioxidant defense systems. Survival rate of such infants has significantly improved due to advances in care involving mechanical ventilation and oxygen supplementation. However, a significant subset of such survivors develops the chronic lung disease, Bronchopulmonary dysplasia (BPD), characterized by enlarged, simplified alveoli and deformed airways. Among a host of factors contributing to the pathogenesis is oxidative damage induced by exposure of the developing lungs to hyperoxia. Recent data indicate that hyperoxia induces aberrant sphingolipid signaling, leading to mitochondrial dysfunction and abnormal reactive oxygen species (ROS) formation (ROS). The role of sphingolipids such as ceramides and sphingosine 1-phosphate (S1P), in the development of BPD emerged in the last decade. Both ceramide and S1P are elevated in tracheal aspirates of premature infants of <32 weeks gestational age developing BPD. This was faithfully reflected in the murine models of hyperoxia and BPD, where there is an increased expression of sphingolipid metabolites both in lung tissue and bronchoalveolar lavage. Treatment of neonatal pups with a sphingosine kinase1 specific inhibitor, PF543, resulted in protection against BPD as neonates, accompanied by improved lung function and reduced airway remodeling as adults. This was accompanied by reduced mitochondrial ROS formation. S1P receptor1 induced by hyperoxia also aggravates BPD, revealing another potential druggable target in this pathway for BPD. In this review we aim to provide a detailed description on the role played by sphingolipid signaling in hyperoxia induced lung injury and BPD.
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Displasia Broncopulmonar/metabolismo , Displasia Broncopulmonar/patología , Lesión Pulmonar/metabolismo , Esfingolípidos/fisiología , Remodelación de las Vías Aéreas (Respiratorias) , Animales , Animales Recién Nacidos , Ceramidas/metabolismo , Modelos Animales de Enfermedad , Humanos , Hiperoxia/metabolismo , Hiperoxia/fisiopatología , Lactante , Recién Nacido , Pulmón/patología , Lesión Pulmonar/patología , Lisofosfolípidos/metabolismo , Metanol/farmacología , Ratones , Estrés Oxidativo/fisiología , Alveolos Pulmonares/metabolismo , Pirrolidinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Esfingolípidos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Sulfonas/farmacologíaRESUMEN
Sphingosine kinase 1 (SPHK1) and the sphingosine-1-phosphate (S1P) signaling pathway have been shown to play a role in pulmonary arterial hypertension (PAH). S1P is an important stimulus for pulmonary artery smooth muscle cell (PASMC) proliferation and pulmonary vascular remodeling. We aimed to examine the specific roles of SPHK1 in PASMCs during pulmonary hypertension (PH) progression. We generated smooth muscle cell-specific, Sphk1-deficient (Sphk1f/f TaglnCre+) mice and isolated Sphk1-deficient PASMCs from SPHK1 knockout mice. We demonstrated that Sphk1f/f TaglnCre+ mice are protected from hypoxia or hypoxia/Sugen-mediated PH, and pulmonary vascular remodeling and that Sphk1-deficient PASMCs are less proliferative compared with ones isolated from wild-type (WT) siblings. S1P or hypoxia activated yes-associated protein 1 (YAP1) signaling by enhancing its translocation to the nucleus, which was dependent on SPHK1 enzymatic activity. Further, verteporfin, a pharmacologic YAP1 inhibitor, attenuated the S1P-mediated proliferation of hPASMCs, hypoxia-mediated PH, and pulmonary vascular remodeling in mice and hypoxia/Sugen-mediated severe PH in rats. Smooth muscle cell-specific SPHK1 plays an essential role in PH via YAP1 signaling, and YAP1 inhibition may have therapeutic potential in treating PH.
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Hipertensión Pulmonar , Fosfotransferasas (Aceptor de Grupo Alcohol) , Proteínas Señalizadoras YAP , Animales , Ratones , Ratas , Proliferación Celular , Células Cultivadas , Hipertensión Pulmonar/metabolismo , Hipoxia/complicaciones , Hipoxia/metabolismo , Miocitos del Músculo Liso/metabolismo , Arteria Pulmonar/metabolismo , Transducción de Señal , Esfingosina/metabolismo , Remodelación Vascular , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Señalizadoras YAP/metabolismoRESUMEN
A history of chronic cigarette smoking is known to increase risk for acute respiratory distress syndrome (ARDS), but the corresponding risks associated with chronic e-cigarette use are largely unknown. The chromosomal fragile site gene, WWOX, is highly susceptible to genotoxic stress from environmental exposures and thus an interesting candidate gene for the study of exposure-related lung disease. Lungs harvested from current versus former/never-smokers exhibited a 47% decrease in WWOX mRNA levels. Exposure to nicotine-containing e-cigarette vapor resulted in an average 57% decrease in WWOX mRNA levels relative to vehicle-treated controls. In separate studies, endothelial (EC)-specific WWOX knockout (KO) versus WWOX flox control mice were examined under ARDS-producing conditions. EC WWOX KO mice exhibited significantly greater levels of vascular leak and histologic lung injury. ECs were isolated from digested lungs of untreated EC WWOX KO mice using sorting by flow cytometry for CD31+ CD45-cells. These were grown in culture, confirmed to be WWOX deficient by RT-PCR and Western blotting, and analyzed by electric cell impedance sensing as well as an FITC dextran transwell assay for their barrier properties during methicillin-resistant Staphylococcus aureus or LPS exposure. WWOX KO ECs demonstrated significantly greater declines in barrier function relative to cells from WWOX flox controls during either methicillin-resistant S. aureus or LPS treatment as measured by both electric cell impedance sensing and the transwell assay. The increased risk for ARDS observed in chronic smokers may be mechanistically linked, at least in part, to lung WWOX downregulation, and this phenomenon may also manifest in the near future in chronic users of e-cigarettes.
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Fumar Cigarrillos/efectos adversos , Regulación hacia Abajo/efectos de los fármacos , Cigarrillo Electrónico a Vapor/efectos adversos , Pulmón/efectos de los fármacos , Nicotina/efectos adversos , Síndrome de Dificultad Respiratoria/inducido químicamente , Oxidorreductasa que Contiene Dominios WW/metabolismo , Animales , Humanos , Pulmón/metabolismo , Masculino , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Ratones , Ratones Endogámicos C57BL , Síndrome de Dificultad Respiratoria/metabolismo , Infecciones Estafilocócicas/metabolismo , Nicotiana/efectos adversos , Productos de Tabaco/efectos adversosRESUMEN
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.
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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
Lysocardiolipin acyltransferase (LYCAT), a cardiolipin (CL)-remodeling enzyme, is crucial for maintaining normal mitochondrial function and vascular development. Despite the well-characterized role for LYCAT in the regulation of mitochondrial dynamics, its involvement in lung cancer, if any, remains incompletely understood. In this study, in silico analysis of TCGA lung cancer data sets revealed a significant increase in LYCAT expression, which was later corroborated in human lung cancer tissues and immortalized lung cancer cell lines via indirect immunofluorescence and immunoblotting, respectively. Stable knockdown of LYCAT in NSCLC cell lines not only reduced CL and increased monolyso-CL levels but also reduced in vivo tumor growth, as determined by xenograft studies in athymic nude mice. Furthermore, blocking LYCAT activity using a LYCAT mimetic peptide attenuated cell migration, suggesting a novel role for LYCAT activity in promoting NSCLC. Mechanistically, the pro-proliferative effects of LYCAT were mediated by an increase in mitochondrial fusion and a G1/S cell cycle transition, both of which are linked to increased cell proliferation. Taken together, these results demonstrate a novel role for LYCAT in promoting NSCLC and suggest that targeting LYCAT expression or activity in NSCLC may provide new avenues for the therapeutic treatment of lung cancer.
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1-Acilglicerol-3-Fosfato O-Aciltransferasa/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Proliferación Celular , Neoplasias Pulmonares/enzimología , Mitocondrias/metabolismo , Proteínas de Neoplasias/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferasa/genética , Células A549 , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Cardiolipinas/genética , Cardiolipinas/metabolismo , Xenoinjertos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Mitocondrias/genética , Proteínas de Neoplasias/genética , Trasplante de NeoplasiasRESUMEN
RATIONALE: The severe acute respiratory syndrome coronavirus 2/coronavirus disease 2019 pandemic has highlighted the serious unmet need for effective therapies that reduce acute respiratory distress syndrome (ARDS) mortality. We explored whether extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a ligand for Toll-like receptor (TLR)4 and a master regulator of innate immunity and inflammation, is a potential ARDS therapeutic target. METHODS: Wild-type C57BL/6J or endothelial cell (EC)-cNAMPT -/- knockout mice (targeted EC NAMPT deletion) were exposed to either a lipopolysaccharide (LPS)-induced ("one-hit") or a combined LPS/ventilator ("two-hit")-induced acute inflammatory lung injury model. A NAMPT-specific monoclonal antibody (mAb) imaging probe (99mTc-ProNamptor) was used to detect NAMPT expression in lung tissues. Either an eNAMPT-neutralising goat polyclonal antibody (pAb) or a humanised monoclonal antibody (ALT-100 mAb) were used in vitro and in vivo. RESULTS: Immunohistochemical, biochemical and imaging studies validated time-dependent increases in NAMPT lung tissue expression in both pre-clinical ARDS models. Intravenous delivery of either eNAMPT-neutralising pAb or mAb significantly attenuated inflammatory lung injury (haematoxylin and eosin staining, bronchoalveolar lavage (BAL) protein, BAL polymorphonuclear cells, plasma interleukin-6) in both pre-clinical models. In vitro human lung EC studies demonstrated eNAMPT-neutralising antibodies (pAb, mAb) to strongly abrogate eNAMPT-induced TLR4 pathway activation and EC barrier disruption. In vivo studies in wild-type and EC-cNAMPT -/- mice confirmed a highly significant contribution of EC-derived NAMPT to the severity of inflammatory lung injury in both pre-clinical ARDS models. CONCLUSIONS: These findings highlight both the role of EC-derived eNAMPT and the potential for biologic targeting of the eNAMPT/TLR4 inflammatory pathway. In combination with predictive eNAMPT biomarker and NAMPT genotyping assays, this offers the opportunity to identify high-risk ARDS subjects for delivery of personalised medicine.
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Lesión Pulmonar Aguda , COVID-19 , Animales , Anticuerpos Monoclonales , Humanos , Ratones , Ratones Endogámicos C57BL , SARS-CoV-2RESUMEN
Osteosarcoma (OS) is a bone malignancy affecting children and adolescents. Retinoblastoma (RB) patients with germline RB1 mutations are susceptible to osteosarcoma in the second decade of their life. Several studies, particularly in mice, have revealed a role for RB1 in osteogenesis. Since, there is species specific difference attributed in retinoblastoma tumorigenesis between mice and human, we assumed, it is worthwhile exploring the role of RB1 in osteogenesis and thus onset of osteosarcoma. In this study, we analyzed the temporal gene expression of the osteogenic markers, tumor suppressor genes and hormone receptors associated with growth spurt during in vitro osteogenesis of mesenchymal stem cells derived from orbital adipose tissue of germline RB patients and compared it with those with wild type RB1 gene. Mesenchymal stem cells with the heterozygous RB1 mutation showed reduced expression of RB1 and other tumor suppressor genes and showed deregulation of osteogenic markers which could be an initial step for the onset of osteosarcoma.
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Neoplasias Óseas/genética , Transformación Celular Neoplásica , Regulación Neoplásica de la Expresión Génica , Mutación , Osteosarcoma/genética , Neoplasias de la Retina/genética , Proteínas de Unión a Retinoblastoma/genética , Ubiquitina-Proteína Ligasas/genética , Adipocitos/metabolismo , Adipocitos/patología , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Adulto , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Diferenciación Celular , Condrocitos/metabolismo , Condrocitos/patología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Femenino , Perfilación de la Expresión Génica , Heterocigoto , Humanos , Masculino , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Órbita/metabolismo , Órbita/patología , Osteogénesis/genética , Osteosarcoma/metabolismo , Osteosarcoma/patología , Cultivo Primario de Células , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Neoplasias de la Retina/metabolismo , Neoplasias de la Retina/patología , Proteínas de Unión a Retinoblastoma/metabolismo , Transducción de Señal , Factor de Transcripción Sp7/genética , Factor de Transcripción Sp7/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Hyperoxia (HO)-induced lung injury contributes to bronchopulmonary dysplasia (BPD) in preterm newborns. Intractable wheezing seen in BPD survivors is associated with airway remodeling (AWRM). Sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) signaling promotes HO-mediated neonatal BPD; however, its role in the sequela of AWRM is not known. We noted an increased concentration of S1P in tracheal aspirates of neonatal infants with severe BPD, and earlier, demonstrated that Sphk1-/- mice showed protection against HO-induced BPD. The role of SPHK1/S1P in promoting AWRM following exposure of neonates to HO was investigated in a murine model. Therapy using PF543, the specific SPHK1 inhibitor, during neonatal HO reduced alveolar simplification followed by reduced AWRM in adult mice. This was associated with reduced airway hyperreactivity to intravenous methacholine. Neonatal HO exposure was associated with increased expression of SPHK1 in lung tissue of adult mice, which was reduced with PF543 therapy in the neonatal stage. This was accompanied by amelioration of HO-induced reduction of E-cadherin in airway epithelium. This may be suggestive of arrested partial epithelial mesenchymal transition (EMT) induced by HO. In vitro studies using human primary airway epithelial cells (HAEpCs) showed that SPHK1 inhibition or deletion restored HO-induced reduction in E-cadherin and reduced formation of mitochondrial reactive oxygen species (mtROS). Blocking mtROS with MitoTempo attenuated HO-induced partial EMT of HAEpCs. These results collectively support a therapeutic role for PF543 in preventing HO-induced BPD in neonates and the long-term sequela of AWRM, thus conferring a long-term protection resulting in improved lung development and function.
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Remodelación de las Vías Aéreas (Respiratorias)/efectos de los fármacos , Displasia Broncopulmonar/tratamiento farmacológico , Hiperoxia/tratamiento farmacológico , Metanol/análogos & derivados , Pirrolidinas/farmacología , Animales , Animales Recién Nacidos , Displasia Broncopulmonar/inducido químicamente , Modelos Animales de Enfermedad , Hiperoxia/inducido químicamente , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Metanol/farmacología , Ratones Noqueados , Fosfotransferasas (Aceptor de Grupo Alcohol)/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , SulfonasRESUMEN
Alveolar epithelial cell (AEC) apoptosis, arising from mitochondrial dysfunction and mitophagy defects, is important in mediating idiopathic pulmonary fibrosis (IPF). Our group established a role for the mitochondrial (mt) DNA base excision repair enzyme, 8-oxoguanine-DNA glycosylase 1 (mtOGG1), in preventing oxidant-induced AEC mtDNA damage and apoptosis and showed that OGG1-deficient mice have increased lung fibrosis. Herein, we determined whether mice overexpressing the mtOGG1 transgene (mtOgg1tg) are protected against lung fibrosis and whether AEC mtOGG1 preservation of mtDNA integrity mitigates phosphatase and tensin homolog-induced putative kinase 1 (PINK1) deficiency and apoptosis. Compared with wild type (WT), mtOgg1tg mice have diminished asbestos- and bleomycin-induced pulmonary fibrosis that was accompanied by reduced lung and AEC mtDNA damage and apoptosis. Asbestos and H2O2 promote the MLE-12 cell PINK1 deficiency, as assessed by reductions in the expression of PINK1 mRNA and mitochondrial protein expression. Compared with WT, Pink1-knockout (Pink1-KO) mice are more susceptible to asbestos-induced lung fibrosis and have increased lung and alveolar type II (AT2) cell mtDNA damage and apoptosis. AT2 cells from Pink1-KO mice and PINK1-silenced (siRNA) MLE-12 cells have increased mtDNA damage that is augmented by oxidative stress. Interestingly, mtOGG1 overexpression attenuates oxidant-induced MLE-12 cell mtDNA damage and apoptosis despite PINK1 silencing. mtDNA damage is increased in the lungs of patients with IPF as compared with controls. Collectively, these findings suggest that mtOGG1 maintenance of AEC mtDNA is crucial for preventing PINK1 deficiency that promotes apoptosis and lung fibrosis. Given the key role of AEC apoptosis in pulmonary fibrosis, strategies aimed at preserving AT2 cell mtDNA integrity may be an innovative target.
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Células Epiteliales Alveolares/efectos de los fármacos , Asbestosis/genética , ADN Glicosilasas/genética , Pulmón/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Proteínas Quinasas/genética , Fibrosis Pulmonar/genética , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/patología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Amianto/administración & dosificación , Asbestosis/etiología , Asbestosis/metabolismo , Asbestosis/patología , Bleomicina/administración & dosificación , Daño del ADN , ADN Glicosilasas/deficiencia , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Femenino , Regulación de la Expresión Génica , Peróxido de Hidrógeno/farmacología , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/metabolismo , Cultivo Primario de Células , Proteínas Quinasas/metabolismo , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Titanio/administración & dosificaciónRESUMEN
BACKGROUND: A new approach targeting aeroallergen sensing in the early events of mucosal immunity could have greater benefit. The CSF1-CSF1R pathway has a critical role in trafficking allergens to regional lymph nodes through activating dendritic cells. Intervention in this pathway could prevent allergen sensitization and subsequent Th2 allergic inflammation. OBJECTIVE: To examine the therapeutic effectiveness of CSF1 and CSF1R inhibition for blocking the dendritic cell function of sensing aeroallergens. METHODS: We adopted a model of chronic asthma induced by a panel of three naturally occurring allergens and novel delivery system of CSF1R inhibitor encapsulated nanoprobe. RESULTS: Selective depletion of CSF1 in airway epithelial cells abolished the production of allergen-reactive IgE, resulting in prevention of new asthma development as well as reversal of established allergic lung inflammation. CDPL-GW nanoprobe containing GW2580, a selective CSF1R inhibitor, showed favorable pharmacokinetics for inhalational treatment and intranasal insufflation delivery of CDPL-GW nanoprobe ameliorated asthma pathologies including allergen-specific serum IgE production, allergic lung and airway inflammation and airway hyper-responsiveness (AHR) with minimal pulmonary adverse reaction. CONCLUSION: The inhibition of the CSF1-CSF1R signaling pathway effectively suppresses sensitization to aeroallergens and consequent allergic lung inflammation in a murine model of chronic asthma. CSF1R inhibition is a promising new target for the treatment of allergic asthma.
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Anisoles/administración & dosificación , Anisoles/farmacología , Asma/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/métodos , Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Factor Estimulante de Colonias de Macrófagos/metabolismo , Pirimidinas/administración & dosificación , Pirimidinas/farmacología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Alérgenos/inmunología , Alérgenos/farmacología , Animales , Asma/inducido químicamente , Modelos Animales de Enfermedad , Femenino , Inmunoglobulina E/biosíntesis , Factor Estimulante de Colonias de Macrófagos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nanoestructuras/administración & dosificación , Compuestos de Amonio Cuaternario/administración & dosificación , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Ácidos Sulfónicos/administración & dosificación , Resultado del TratamientoRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease of unknown etiology characterized by distorted distal lung architecture, inflammation, and fibrosis. The molecular mechanisms involved in the pathophysiology of IPF are incompletely defined. Several lung cell types including alveolar epithelial cells, fibroblasts, monocyte-derived macrophages, and endothelial cells have been implicated in the development and progression of fibrosis. Regardless of the cell types involved, changes in gene expression, disrupted glycolysis, and mitochondrial oxidation, dysregulated protein folding, and altered phospholipid and sphingolipid metabolism result in activation of myofibroblast, deposition of extracellular matrix proteins, remodeling of lung architecture and fibrosis. Lipid mediators derived from phospholipids, sphingolipids, and polyunsaturated fatty acids play an important role in the pathogenesis of pulmonary fibrosis and have been described to exhibit pro- and anti-fibrotic effects in IPF and in preclinical animal models of lung fibrosis. This review describes the current understanding of the role and signaling pathways of prostanoids, lysophospholipids, and sphingolipids and their metabolizing enzymes in the development of lung fibrosis. Further, several of the lipid mediators and enzymes involved in their metabolism are therapeutic targets for drug development to treat IPF.
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Redes Reguladoras de Genes , Fibrosis Pulmonar Idiopática/metabolismo , Metabolismo de los Lípidos , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Glucólisis , Humanos , Fibrosis Pulmonar Idiopática/genética , Transducción de SeñalRESUMEN
The sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) signaling axis is emerging as a key player in the development of idiopathic pulmonary fibrosis (IPF) and bleomycin (BLM)-induced lung fibrosis in mice. Recent evidence implicates the involvement of the Hippo/Yes-associated protein (YAP) 1 pathway in lung diseases, including IPF, but its plausible link to the SPHK1/S1P signaling pathway is unclear. Herein, we demonstrate the increased co-localization of YAP1 with the fibroblast marker FSP1 in the lung fibroblasts of BLM-challenged mice, and the genetic deletion of Sphk1 in mouse lung fibroblasts (MLFs) reduced YAP1 localization in fibrotic foci. The PF543 inhibition of SPHK1 activity in mice attenuated YAP1 co-localization with FSP1 in lung fibroblasts. In vitro, TGF-ß stimulated YAP1 translocation to the nucleus in primary MLFs, and the deletion of Sphk1 or inhibition with PF543 attenuated TGF-ß-mediated YAP1 nuclear localization. Moreover, the PF543 inhibition of SPHK1, or the verteporfin inhibition of YAP1, decreased the TGF-ß- or BLM-induced mitochondrial reactive oxygen species (mtROS) in human lung fibroblasts (HLFs) and the expression of fibronectin (FN) and alpha-smooth muscle actin (α-SMA). Furthermore, scavenging mtROS with MitoTEMPO attenuated the TGF-ß-induced expression of FN and α-SMA. The addition of the S1P antibody to HLFs reduced TGF-ß- or S1P-mediated YAP1 activation, mtROS, and the expression of FN and α-SMA. These results suggest a role for SPHK1/S1P signaling in TGF-ß-induced YAP1 activation and mtROS generation, resulting in fibroblast activation, a critical driver of pulmonary fibrosis.