RESUMEN
The 18-kDa isoform of basic fibroblast growth factor (bFGF/FGF2) lacks a conventional signal peptide sequence and is exported by a novel membrane-associated transport pathway. Extracellular vesicles (EVs) are increasingly recognized as mediators of intercellular communication in the lung, and our prior work demonstrates that EVs carry cargo that contributes to hyperoxic lung injury and are biomarkers for bronchopulmonary dysplasia. We used primary human bronchial epithelial (HBE), pulmonary artery endothelial (HPAE), and fibroblast (HNF) cells to determine whether FGF2 was secreted in EVs. EVs were isolated by ultracentrifugation from HBE, HPAE, and HNF exposed to either normoxia or hyperoxia, followed by nanoparticle tracking analysis and electron microscopy. Hyperoxia exposure increased the total EV number. All three cell types released FGF2-18kDa both directly into the extracellular environment (secretome), as well as in EVs. HBE released more FGF2-18kDa in EVs during hyperoxia, and these were internalized and localized to both nuclei and cytoplasm of recipient cells. By co-immunoprecipitation, we identified potential binding partners of FGF2-18kDa in the nuclei, including histone 1.2 (H1.2) binding protein, that may mediate downstream effects that do not involve FGF2 binding to cell surface receptors. FGF2-18kDa interaction with H1.2 binding protein may indicate a mechanism by which FGF2 secreted in EVs modulates cellular processes. FGF2 was also found to increase angiogenesis by Matrigel assay. Further studies are necessary to determine the biological relevance of FGF2 in EVs as modulators of lung injury and disease.NEW & NOTEWORTHY We found that multiple lung cell types release basic fibroblast growth factor (FGF2)-18kDa both directly into the extracellular environment (secretome), as well as in extracellular vesicles (EVs). Bronchial epithelial cells released more FGF2-18kDa in EVs during hyperoxia, which could be internalized rapidly by recipient cells. We also identified potential binding partners of FGF2-18kDa in nuclei that may mediate downstream effects that do not involve FGF2 binding to cell surface receptors. We also confirmed a potential angiogenic role for FGF2-18kDa.
Asunto(s)
Vesículas Extracelulares , Factor 2 de Crecimiento de Fibroblastos , Pulmón , Humanos , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Vesículas Extracelulares/metabolismo , Pulmón/metabolismo , Pulmón/patología , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Células Endoteliales/metabolismo , Células Cultivadas , Hiperoxia/metabolismo , Hiperoxia/patología , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Bronquios/metabolismo , Bronquios/patologíaRESUMEN
BACKGROUND: MicroRNA (miR) are small conserved RNA that regulate gene expression post-transcription. Previous genome-wide analysis studies in preterm infants indicate that pathways of miR 219-5p are important in infants with Bronchopulmonary Dysplasia (BPD). METHODS: Here we report a prospective cohort study of extremely preterm neonates wherein infants diagnosed with severe BPD expressed increased airway miR-219-5p and decreased platelet derived growth factor receptor alpha (PDGFR-α), a target of mir-219-5p and a key regulator of alveolarization, compared to post-conception age-matched term infants. RESULTS: miR-219-5p was highly expressed in the pulmonary epithelial lining in lungs of infants with BPD by in situ hybridization of human infant lungs. In both in vitro and in vivo (mouse) models of BPD, miR-219-5p was increased on exposure to hyperoxia compared with the normoxia control, with a complementary decrease of PDGFR-α. To further confirm the target relationship between miR-219 and PDGFR-α, pulmonary epithelial cells (MLE12) and lung primary fibroblasts were treated with a mimic of miR-219-5p and a locked nucleic acid (LNA) based inhibitor of miR-219-5p. In comparison with the control group, the level of miR-219 increased significantly after miR-219 mimic treatment, while the level of PDGFR-α declined markedly. LNA exposure increased PDGFR-α. Moreover, in BPD mouse model, over-expression of miR-219-5p inhibited alveolar development, indicated by larger alveolar spaces accompanied by reduced septation. CONCLUSIONS: Taken together, our results demonstrate that increased miR-219-5p contributes to the pathogenesis of BPD by targeting and reducing PDGFR-α. The use of specific miRNA antagonists may be a therapeutic strategy for preventing the development of BPD.
Asunto(s)
Displasia Broncopulmonar/metabolismo , MicroARNs/biosíntesis , Alveolos Pulmonares/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Animales , Animales Recién Nacidos , Displasia Broncopulmonar/patología , Displasia Broncopulmonar/terapia , Estudios de Cohortes , Presión de las Vías Aéreas Positiva Contínua/métodos , Humanos , Recién Nacido , Recien Nacido Prematuro/metabolismo , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Estudios Prospectivos , Alveolos Pulmonares/patologíaRESUMEN
Cyclooxygenase-2 (COX-2/PTGS2) mediates hyperoxia-induced impairment of lung development in newborn animals and is increased in the lungs of human infants with bronchopulmonary dysplasia (BPD). COX-2 catalyzes the production of cytoprotective prostaglandins, such as prostacyclin (PGI2), as well as proinflammatory mediators, such as thromboxane A2. Our objective was to determine whether iloprost, a synthetic analog of PGI2, would attenuate hyperoxia effects in the newborn mouse lung. To test this hypothesis, newborn C57BL/6 mice along with their dams were exposed to normoxia (21% O2) or hyperoxia (85% O2) from 4 to 14 days of age in combination with daily intraperitoneal injections of either iloprost 200 µg·kg-1·day-1, nimesulide (selective COX-2 antagonist) 100 mg·kg-1·day-1, or vehicle. Alveolar development was estimated by radial alveolar counts and mean linear intercepts. Lung function was determined on a flexiVent, and multiple cytokines and myeloperoxidase (MPO) were quantitated in lung homogenates. Lung vascular and microvascular morphometry was performed, and right ventricle/left ventricle ratios were determined. We determined that iloprost (but not nimesulide) administration attenuated hyperoxia-induced inhibition of alveolar development and microvascular density in newborn mice. Iloprost and nimesulide both attenuated hyperoxia-induced, increased lung resistance but did not improve lung compliance that was reduced by hyperoxia. Iloprost and nimesulide reduced hyperoxia-induced increases in MPO and some cytokines (IL-1ß and TNF-α) but not others (IL-6 and KC/Gro). There were no changes in pulmonary arterial wall thickness or right ventricle/left ventricle ratios. We conclude that iloprost improves lung development and reduces lung inflammation in a newborn mouse model of BPD.
Asunto(s)
Displasia Broncopulmonar/tratamiento farmacológico , Hiperoxia/fisiopatología , Iloprost/farmacología , Neumonía/prevención & control , Alveolos Pulmonares/efectos de los fármacos , Animales , Animales Recién Nacidos , Displasia Broncopulmonar/etiología , Displasia Broncopulmonar/metabolismo , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos C57BL , Neumonía/etiología , Neumonía/metabolismo , Embarazo , Alveolos Pulmonares/irrigación sanguínea , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Vasodilatadores/farmacologíaRESUMEN
RATIONALE: Vascular endothelial mitochondrial dysfunction contributes to the pathogenesis of several oxidant stress-associated disorders. Oxidant stress is a major contributor to the pathogenesis of bronchopulmonary dysplasia (BPD), a chronic lung disease of prematurity that often leads to sequelae in adult survivors. OBJECTIVES: This study was conducted to identify whether differences in mitochondrial bioenergetic function and oxidant generation in human umbilical vein endothelial cells (HUVECs) obtained from extremely preterm infants were associated with risk for BPD or death before 36 weeks postmenstrual age. METHODS: HUVEC oxygen consumption and superoxide and hydrogen peroxide generation were measured in 69 infants. MEASUREMENTS AND MAIN RESULTS: Compared with HUVECs from infants who survived without BPD, HUVECs obtained from infants who developed BPD or died had a lower maximal oxygen consumption rate (mean ± SEM, 107 ± 8 vs. 235 ± 22 pmol/min/30,000 cells; P < 0.001), produced more superoxide after exposure to hyperoxia (mean ± SEM, 89,807 ± 16,616 vs. 162,706 ± 25,321 MitoSOX Red fluorescence units; P < 0.05), and released more hydrogen peroxide into the supernatant after hyperoxia exposure (mean ± SEM, 1,879 ± 278 vs. 842 ± 119 resorufin arbitrary fluorescence units; P < 0.001). CONCLUSIONS: Our results indicating that endothelial cells of premature infants who later develop BPD or die have impaired mitochondrial bioenergetic capacity and produce more oxidants at birth suggest that the vascular endothelial mitochondrial dysfunction seen at birth in these infants persists through their postnatal life and contributes to adverse pulmonary outcomes and increased early mortality.
Asunto(s)
Displasia Broncopulmonar/etiología , Displasia Broncopulmonar/mortalidad , Enfermedades Mitocondriales/etiología , Enfermedades Mitocondriales/mortalidad , Respiración Artificial/efectos adversos , Enfermedades Vasculares/etiología , Enfermedades Vasculares/mortalidad , Displasia Broncopulmonar/fisiopatología , Femenino , Humanos , Recien Nacido Extremadamente Prematuro/fisiología , Recién Nacido , Recien Nacido Prematuro/fisiología , Masculino , Enfermedades Mitocondriales/fisiopatología , Estados Unidos , Enfermedades Vasculares/fisiopatologíaRESUMEN
MicroRNAs (miRs) are small conserved RNA that regulate gene expression. Bioinformatic analysis of miRNA profiles during mouse lung development indicated a role for multiple miRNA, including miRNA-489. miR-489 increased on completion of alveolar septation [postnatal day 42 (P42)], associated with decreases in its conserved target genes insulin-like growth factor-1 (Igf1) and tenascin C (Tnc). We hypothesized that dysregulation of miR-489 and its target genes Igf1 and Tnc contribute to hyperoxia-induced abnormal lung development. C57BL/6 mice were exposed to normoxia (21%) or hyperoxia (85% O2) from P4 to P14, in combination with intranasal locked nucleic acid against miR-489 to inhibit miR-489, cytomegalovirus promoter (pCMV)-miR-489 to overexpress miR-489, or empty vector. Hyperoxia reduced miR-489 and increased Igf1 and Tnc. Locked nucleic acid against miR-489 improved lung development during hyperoxia and did not alter it during normoxia, whereas miR-489 overexpression inhibited lung development during normoxia. The 3' untranslated region in vitro reporter studies confirmed Igf1 and Tnc as targets of miR-489. While miR-489 was of epithelial origin and present in exosomes, its targets Igf1 and Tnc were produced by fibroblasts. Infants with bronchopulmonary dysplasia (BPD) had reduced lung miR-489 and increased Igf1 and Tnc compared with normal preterm or term infants. These results suggest increased miR-489 is an inhibitor of alveolar septation. During hyperoxia or BPD, reduced miR-489 and increased Igf1 and Tnc may be inadequate attempts at compensation. Further inhibition of miR-489 may permit alveolar septation to proceed. The use of specific miRNA antagonists or agonists may be a therapeutic strategy for inhibited alveolarization, such as in BPD.
Asunto(s)
Hiperoxia/metabolismo , MicroARNs/genética , Alveolos Pulmonares/metabolismo , Animales , Animales Recién Nacidos , Displasia Broncopulmonar/metabolismo , Proliferación Celular/genética , Proliferación Celular/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Humanos , Ratones Endogámicos C57BLAsunto(s)
Displasia Broncopulmonar , Humanos , Lactante , Recién Nacido , Recien Nacido Prematuro , Pulmón , MitocondriasRESUMEN
Hyperoxia-induced inflammation contributes significantly to developmental lung injury and bronchopulmonary dysplasia (BPD) in preterm infants. Platelet activating factor (PAF) is known to be a major driver of inflammation in lung diseases such as asthma and pulmonary fibrosis, but its role in BPD has not been previously investigated. Therefore, to determine whether PAF signaling independently modulates neonatal hyperoxic lung injury and BPD pathogenesis, lung structure was assessed in 14 day-old C57BL/6 wild-type (WT) and PAF receptor knockout (PTAFR KO) mice that were exposed to 21% (normoxia) or 85% O 2 (hyperoxia) from postnatal day 4. Lung morphometry showed that PTAFR KO mice had attenuated hyperoxia-induced alveolar simplification when compared to WT mice. Functional analysis of gene expression data from hyperoxia-exposed vs. normoxia-exposed lungs of WT and PTAFR KO showed that the most upregulated pathways were the hypercytokinemia/hyperchemokinemia pathway in WT mice, NAD signaling pathway in PTAFR KO mice, and agranulocyte adhesion and diapedesis as well as other pro-fibrotic pathways such as tumor microenvironment and oncostatin-M signaling in both mice strains, indicating that PAF signaling may contribute to inflammation but may not be a significant mediator of fibrotic processes during hyperoxic neonatal lung injury. Gene expression analysis also indicated increased expression of pro-inflammatory genes such as CXCL1, CCL2 and IL-6 in the lungs of hyperoxia-exposed WT mice and metabolic regulators such as HMGCS2 and SIRT3 in the lungs of PTAFR KO mice, suggesting that PAF signaling may modulate BPD risk through changes in pulmonary inflammation and/or metabolic reprogramming in preterm infants.
RESUMEN
Oxidative stress is an important contributor to bronchopulmonary dysplasia (BPD), a form of chronic lung disease that is the most common morbidity in very preterm infants. Mitochondrial functional differences due to inherited and acquired mutations influence the pathogenesis of disorders in which oxidative stress plays a critical role. We previously showed using mitochondrial-nuclear exchange (MNX) mice that mitochondrial DNA (mtDNA) variations modulate hyperoxia-induced lung injury severity in a model of BPD. In this study, we studied the effects of mtDNA variations on mitochondrial function including mitophagy in alveolar epithelial cells (AT2) from MNX mice. We also investigated oxidant and inflammatory stress as well as transcriptomic profiles in lung tissue in mice and expression of proteins such as PINK1, Parkin and SIRT3 in infants with BPD. Our results indicate that AT2 from mice with C57 mtDNA had decreased mitochondrial bioenergetic function and inner membrane potential, increased mitochondrial membrane permeability and were exposed to higher levels of oxidant stress during hyperoxia compared to AT2 from mice with C3H mtDNA. Lungs from hyperoxia-exposed mice with C57 mtDNA also had higher levels of pro-inflammatory cytokines compared to lungs from mice with C3H mtDNA. We also noted changes in KEGG pathways related to inflammation, PPAR and glutamatergic signaling, and mitophagy in mice with certain mito-nuclear combinations but not others. Mitophagy was decreased by hyperoxia in all mice strains, but to a greater degree in AT2 and neonatal mice lung fibroblasts from hyperoxia-exposed mice with C57 mtDNA compared to C3H mtDNA. Finally, mtDNA haplogroups vary with ethnicity, and Black infants with BPD had lower levels of PINK1, Parkin and SIRT3 expression in HUVEC at birth and tracheal aspirates at 28 days of life when compared to White infants with BPD. These results indicate that predisposition to neonatal lung injury may be modulated by variations in mtDNA and mito-nuclear interactions need to be investigated to discover novel pathogenic mechanisms for BPD.
RESUMEN
We have previously shown that inhibition of transforming growth factor-ß (TGF-ß) signaling attenuates hypoxia-induced inhibition of alveolar development and abnormal pulmonary vascular remodeling in the newborn mice and that endothelin-A receptor (ETAR) antagonists prevent and reverse the vascular remodeling. The current study tested the hypothesis that inhibition of TGF-ß signaling attenuates endothelin-1 (ET-1) expression and thereby reduces effects of hypoxia on the newborn lung. C57BL/6 mice were exposed from birth to 2 wk of age to either air or hypoxia (12% O(2)) while being given either BQ610 (ETAR antagonist), BQ788 (ETBR antagonist), 1D11 (TGF-ß neutralizing antibody), or vehicle. Lung function and development and TGF-ß and ET-1 synthesis were assessed. Hypoxia inhibited alveolar development, decreased lung compliance, and increased lung resistance. These effects were associated with increased TGF-ß synthesis and signaling and increased ET-1 synthesis. BQ610 (but not BQ788) improved lung function, without altering alveolar development or increased TGF-ß signaling in hypoxia-exposed animals. Inhibition of TGF-ß signaling reduced ET-1 in vivo, which was confirmed in vitro in mouse pulmonary endothelial, fibroblast, and epithelial cells. ETAR blockade improves function but not development of the hypoxic newborn lung. Reduction of ET-1 via inhibition of TGF-ß signaling indicates that TGF-ß is upstream of ET-1 during hypoxia-induced signaling in the newborn lung.
Asunto(s)
Endotelina-1/fisiología , Hipoxia/metabolismo , Pulmón/fisiopatología , Transducción de Señal , Factor de Crecimiento Transformador beta/fisiología , Animales , Animales Recién Nacidos , Células Cultivadas , Células Endoteliales/metabolismo , Antagonistas de los Receptores de la Endotelina A , Antagonistas de los Receptores de la Endotelina B , Endotelina-1/genética , Endotelina-1/metabolismo , Endotelio Vascular/citología , Expresión Génica , Pulmón/crecimiento & desarrollo , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Oligopéptidos/farmacología , Piperidinas/farmacología , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Hypoxia enhances transforming growth factor-ß (TGF-ß) signaling, inhibiting alveolar development and causing abnormal pulmonary arterial remodeling in the newborn lung. We hypothesized that, during chronic hypoxia, reduced peroxisome proliferator-activated receptor-γ (PPAR-γ) signaling may contribute to, or be caused by, excessive TGF-ß signaling. To determine whether PPAR-γ was reduced during hypoxia, C57BL/6 mice were exposed to hypoxia from birth to 2 wk and evaluated for PPAR-γ mRNA and protein. To determine whether rosiglitazone (RGZ, a PPAR-γ agonist) supplementation attenuated the effects of hypoxia, mice were exposed to air or hypoxia from birth to 2 wk in combination with either RGZ or vehicle, and measurements of lung histology, function, parameters related to TGF-ß signaling, and collagen content were made. To determine whether excessive TGF-ß signaling reduced PPAR-γ, mice were exposed to air or hypoxia from birth to 2 wk in combination with either TGF-ß-neutralizing antibody or vehicle, and PPAR-γ signaling was evaluated. We observed that hypoxia reduced PPAR-γ mRNA and protein, in association with impaired alveolarization, increased TGF-ß signaling, reduced lung compliance, and increased collagen. RGZ increased PPAR-γ signaling, with improved lung development and compliance in association with reduced collagen and TGF-ß signaling. However, no reduction was noted in hypoxia-induced pulmonary vascular remodeling. Inhibition of hypoxia-enhanced TGF-ß signaling increased PPAR-γ signaling. These results suggest that hypoxia-induced inhibition of lung development is associated with a mutually antagonistic relationship between reduced PPAR-γ and increased TGF-ß signaling. PPAR-γ agonists may be of potential therapeutic significance in attenuating TGF-ß signaling and improving alveolar development.
Asunto(s)
Hipoxia/patología , Pulmón/efectos de los fármacos , Pulmón/crecimiento & desarrollo , PPAR gamma/agonistas , Tiazolidinedionas/farmacología , Remodelación de las Vías Aéreas (Respiratorias)/efectos de los fármacos , Animales , Animales Recién Nacidos , Anticuerpos Neutralizantes/farmacología , Enfermedad Crónica , Colágeno/metabolismo , Hipertrofia Ventricular Derecha/complicaciones , Hipertrofia Ventricular Derecha/fisiopatología , Hipoxia/complicaciones , Hipoxia/fisiopatología , Pulmón/patología , Pulmón/fisiopatología , Ratones , Ratones Endogámicos C57BL , PPAR gamma/metabolismo , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Alveolos Pulmonares/fisiopatología , Rosiglitazona , Transducción de Señal/efectos de los fármacos , Tiazolidinedionas/administración & dosificación , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The polyphenol quercetin (Quer) represses expression of the cardiovascular disease risk factor plasminogen activator inhibitor-1 (PAI-1) in cultured endothelial cells (ECs). Transfection of PAI-1 promoter-luciferase reporter deletion constructs identified a 251-bp fragment (nucleotides -800 to -549) responsive to Quer. Two E-box motifs (CACGTG), at map positions -691 (E-box1) and -575 (E-box2), are platforms for occupancy by several members of the c-MYC family of basic helix-loop-helix leucine zipper (bHLH-LZ) proteins. Promoter truncation and electrophoretic mobility shift/supershift analyses identified upstream stimulatory factor (USF)-1 and USF-2 as E-box1/E-box2 binding factors. ECs co-transfected with a 251 bp PAI-1 promoter fragment containing the two E-box motifs (p251/luc) and a USF-2 expression vector (pUSF-2/pcDNA) exhibited reduced luciferase activity versus p251/luc alone. Overexpression of USF-2 decreased, while transfection of a dominant-negative USF construct increased, EC growth consistent with the known anti-proliferative properties of USF proteins. Quer-induced decreases in PAI-1 expression and reduced cell proliferation may contribute, at least in part, to the cardioprotective benefit associated with daily intake of polyphenols.
Asunto(s)
Células Endoteliales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Inhibidor 1 de Activador Plasminogénico/genética , Quercetina/farmacología , Factores Estimuladores hacia 5'/fisiología , Cardiotónicos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Elementos E-Box , Células Endoteliales/citología , Humanos , Leucina Zippers/genética , Fragmentos de Péptidos , Inhibidor 1 de Activador Plasminogénico/biosíntesisRESUMEN
Premature infants are at high risk for developing bronchopulmonary dysplasia (BPD), characterized by chronic inflammation and inhibition of lung development, which we have recently identified as being modulated by microRNAs (miRNAs) and alterations in the airway microbiome. Exosomes and exosomal miRNAs may regulate cell differentiation and tissue and organ development. We discovered that tracheal aspirates from infants with severe BPD had increased numbers of, but smaller, exosomes compared with term controls. Similarly, bronchoalveolar lavage fluid from hyperoxia-exposed mice (an animal model of BPD) and supernatants from hyperoxia-exposed human bronchial epithelial cells (in vitro model of BPD) had increased exosomes compared with air controls. Next, in a prospective cohort study of tracheal aspirates obtained at birth from extremely preterm infants, utilizing independent discovery and validation cohorts, we identified unbiased exosomal miRNA signatures predictive of severe BPD. The strongest signal of reduced miR-876-3p in BPD-susceptible compared with BPD-resistant infants was confirmed in the animal model and in vitro models of BPD. In addition, based on our recent discovery of increased Proteobacteria in the airway microbiome being associated with BPD, we developed potentially novel in vivo and in vitro models for BPD combining Proteobacterial LPS and hyperoxia exposure. Addition of LPS led to a larger reduction in exosomal miR 876-3p in both hyperoxia and normoxia compared with hyperoxia alone, thus indicating a potential mechanism by which alterations in microbiota can suppress miR 876-3p. Gain of function of miR 876-3p improved the alveolar architecture in the in vivo BPD model, demonstrating a causal link between miR 876-3p and BPD. In summary, we provide evidence for the strong predictive biomarker potential of miR 876-3p in severe BPD. We also provide insights on the pathogenesis of neonatal lung disease, as modulated by hyperoxia and microbial product-induced changes in exosomal miRNA 876-3p, which could be targeted for future therapeutic development.
Asunto(s)
Células Epiteliales Alveolares/inmunología , Displasia Broncopulmonar/diagnóstico , Exosomas/metabolismo , Recien Nacido Extremadamente Prematuro/inmunología , MicroARNs/metabolismo , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/microbiología , Animales , Animales Recién Nacidos , Biomarcadores/metabolismo , Líquido del Lavado Bronquioalveolar/citología , Displasia Broncopulmonar/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular , Modelos Animales de Enfermedad , Exosomas/genética , Exosomas/inmunología , Femenino , Humanos , Hiperoxia/inmunología , Recien Nacido con Peso al Nacer Extremadamente Bajo/inmunología , Recién Nacido , Lipopolisacáridos/inmunología , Masculino , Ratones , MicroARNs/genética , MicroARNs/inmunología , Microbiota/inmunología , Pronóstico , Estudios Prospectivos , Proteobacteria/inmunología , Índice de Severidad de la EnfermedadRESUMEN
Epidemiologic data have indicated that the intake of polyphenols is inversely associated with mortality from cardiovascular disease. Mitogen-activated protein kinases (MAPKs) are ubiquitous signaling proteins that have been associated with gene regulation. This study determined whether polyphenols (catechin and quercetin) activated kinase-signaling cascades that suppress PAI-1 expression and whether this suppression is at the transcription level in human coronary artery endothelial cells (ECs) remains unresolved. ECs were incubated in the absence/presence of polyphenols and RNA and protein were analyzed by real-time PCR and Western blot analysis. MAPKs were analyzed using antibodies to active form of p38, JNK, and ERK1/2. ECs were transiently transfected with a 1.1-kb PAI-1 promoter (pPAI110/luc) and promoter activity were assays after treatment with polyphenols. Catechin and quercetin decreased EC PAI-1 mRNA in a time- and dose-dependent manner, reaching a maximum at 4 and 2 h, respectively. These polyphenols activated EC p38 and ERK1/2 within 2.5 and 5 min, respectively, while maximal JNK activation occurred at 10-15 min. An inhibitor of p38 MAPK had no effect on polyphenol-induced repression of PAI-1. Inhibitors of ERK or JNK prevented polyphenol repression of EC PAI-1 gene expression. Exposing ECs transiently transfected with pPAI110/luc to polyphenols decreased promoter activity 50%. Polyphenols repress EC PAI-1 expression, in part, by activating ERK and JNK signaling pathways and this repression is at transcriptional levels. Thus MAPK seem to play an important role in polyphenol-induce repression of PAI-1 expression in ECs.