RÉSUMÉ
Pulmonary Arterial Hypertension (PAH) is overrepresented in People Living with Human Immunodeficiency Virus (PLWH). HIV protein gp120 plays a key role in the pathogenesis of HIV-PAH. Genetic changes in HIV gp120 determine viral interactions with chemokine receptors; specifically, HIV-X4 viruses interact with CXCR4 while HIV-R5 interact with CCR5 co-receptors. Herein, we leveraged banked samples from patients enrolled in the NIH Lung HIV studies and used bioinformatic analyses to investigate whether signature sequences in HIV-gp120 that predict tropism also predict PAH. Further biological assays were conducted in pulmonary endothelial cells in vitro and in HIV-transgenic rats. We found that significantly more persons living with HIV-PAH harbor HIV-X4 variants. Multiple HIV models showed that recombinant gp120-X4 as well as infectious HIV-X4 remarkably increase arachidonate 5-lipoxygenase (ALOX5) expression. ALOX5 is essential for the production of leukotrienes; we confirmed that leukotriene levels are increased in bronchoalveolar lavage fluid of HIV-infected patients. This is the first report associating HIV-gp120 genotype to a pulmonary disease phenotype, as we uncovered X4 viruses as potential agents in the pathophysiology of HIV-PAH. Altogether, our results allude to the supplementation of antiretroviral therapy with ALOX5 antagonists to rescue patients with HIV-X4 variants from fatal PAH.
Sujet(s)
Arachidonate 5-lipoxygenase/métabolisme , Infections à VIH/complications , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/génétique , Poumon/métabolisme , Hypertension artérielle pulmonaire/complications , Tropisme viral/génétique , Adulte , Animaux , Agents antiVIH/usage thérapeutique , Cellules cultivées , Études de cohortes , Modèles animaux de maladie humaine , Cellules endothéliales/métabolisme , Femelle , Génotype , Protéine d'enveloppe gp120 du VIH/génétique , Infections à VIH/traitement médicamenteux , Infections à VIH/virologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/métabolisme , Humains , Mâle , Adulte d'âge moyen , Phénotype , Hypertension artérielle pulmonaire/virologie , Artère pulmonaire/cytologie , Rats , Rats de lignée F344 , Rats transgéniques , Récepteurs CXCR4/métabolismeRÉSUMÉ
Cystic fibrosis (CF) is a progressive multiorgan autosomal recessive disease with devastating impact on the lungs caused by derangements of the CF transmembrane conductance regulator (CFTR) gene. Morbidity and mortality are caused by the triad of impaired mucociliary clearance, microbial infections and chronic inflammation. Pseudomonas aeruginosa is the main respiratory pathogen in individuals with CF infecting most patients in later stages. Despite its recognized clinical impact, molecular mechanisms that underlie P. aeruginosa pathogenesis and the host response to P. aeruginosa infection remain incompletely understood. The nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) γ (PPARγ), has shown to be reduced in CF airways. In the present study, we sought to investigate the upstream mechanisms repressing PPARγ expression and its impact on airway epithelial host defense. Endoplasmic reticulum-stress (ER-stress) triggered unfolded protein response (UPR) activated by misfolded CFTR and P. aeruginosa infection contributed to attenuated expression of PPARγ. Specifically, the protein kinase RNA (PKR)-like ER kinase (PERK) signaling pathway led to the enhanced expression of the CCAAT-enhancer-binding-protein homologous protein (CHOP). CHOP induction led to the repression of PPARγ expression. Mechanistically, we showed that CHOP induction mediated PPARγ attenuation, impacted the innate immune function of normal and ∆F508 primary airway epithelial cells by reducing expression of antimicrobial peptide (AMP) and paraoxanse-2 (PON-2), as well as enhancing IL-8 expression. Furthermore, mitochondrial reactive oxygen species production (mt-ROS) and ER-stress positive feedforward loop also dysregulated mitochondrial bioenergetics. Additionally, our findings implicate that PPARγ agonist pioglitazone (PIO) has beneficial effect on the host at the multicellular level ranging from host defense to mitochondrial re-energization.
Sujet(s)
Mucoviscidose/métabolisme , Récepteur PPAR gamma/métabolisme , Infections à Pseudomonas/métabolisme , Pseudomonas aeruginosa/physiologie , Réponse aux protéines mal repliées , Cellules A549 , Aryldialkylphosphatase/métabolisme , Mucoviscidose/complications , Mucoviscidose/microbiologie , Stress du réticulum endoplasmique , Cellules épithéliales/métabolisme , Interactions hôte-pathogène , Humains , Immunité innée , Interleukine-8/métabolisme , Mitochondries/métabolisme , Récepteur PPAR gamma/agonistes , Pioglitazone , Infections à Pseudomonas/immunologie , Facteur de transcription CHOP/métabolisme , bêta-Défensines/métabolismeRÉSUMÉ
Recent studies have implicated autophagy in several inflammatory diseases involving aberrant endothelial cell (EC) responses, such as acute lung injury (ALI). However, the mechanistic basis for a role of autophagy in EC inflammation and permeability remain poorly understood. In this study, we impaired autophagy by silencing the essential Beclin1 autophagy gene in human pulmonary artery EC. This resulted in reduced expression of proinflammatory genes in response to thrombin, a procoagulant and proinflammatory mediator whose concentration is elevated in many diseases including sepsis and ALI. These (Beclin1-depleted) cells also displayed a marked decrease in NF-κB activity secondary to impaired DNA binding of RelA/p65 in the nucleus, but exhibited normal IκBα degradation in the cytosol. Further analysis showed that Beclin1 knockdown was associated with impaired RelA/p65 translocation to the nucleus. Additionally, Beclin1 knockdown attenuated thrombin-induced phosphorylation of RelA/p65 at Ser536, a critical event necessary for the transcriptional activity of RelA/p65. Beclin1 silencing also protected against thrombin-induced EC barrier disruption by preventing the loss of VE-cadherin at adherens junctions. Moreover, Beclin1 knockdown reduced thrombin-induced phosphorylation/inactivation of actin depolymerizing protein Cofilin1 and thereby actin stress fiber formation required for EC permeability as well as RelA/p65 nuclear translocation. Together, these data identify Beclin1 as a novel mechanistic link between autophagy and EC dysfunction (inflammation and permeability).
Sujet(s)
Jonctions adhérentes/métabolisme , Autophagie/génétique , Bécline-1/métabolisme , Cellules endothéliales/métabolisme , Facteur de transcription RelA/métabolisme , Autophagie/effets des médicaments et des substances chimiques , Bécline-1/génétique , Noyau de la cellule/métabolisme , Cellules cultivées , Cofiline-1/métabolisme , ADN/métabolisme , Techniques de knock-down de gènes , Humains , Inflammation/métabolisme , Inhibiteur alpha de NF-KappaB/métabolisme , Phosphorylation/effets des médicaments et des substances chimiques , Phosphorylation/génétique , Artère pulmonaire/cytologie , Transduction du signal/effets des médicaments et des substances chimiques , Transduction du signal/génétique , Thrombine/pharmacologie , TransfectionRÉSUMÉ
Pulmonary hypertension (PH) is a progressive disorder that causes significant morbidity and mortality despite existing therapies. PH pathogenesis is characterized by metabolic derangements that increase pulmonary artery smooth muscle cell (PASMC) proliferation and vascular remodeling. PH-associated decreases in peroxisome proliferator-activated receptor γ (PPARγ) stimulate PASMC proliferation, and PPARγ in coordination with PPARγ coactivator 1α (PGC1α) regulates mitochondrial gene expression and biogenesis. To further examine the impact of decreases in PPARγ expression on human PASMC (HPASMC) mitochondrial function, we hypothesized that depletion of either PPARγ or PGC1α perturbs mitochondrial structure and function to stimulate PASMC proliferation. To test this hypothesis, HPASMCs were exposed to hypoxia and treated pharmacologically with the PPARγ antagonist GW9662 or with siRNA against PPARγ or PGC1α for 72 hours. HPASMC proliferation (cell counting), target mRNA levels (qRT-PCR), target protein levels (Western blotting), mitochondria-derived H2O2 (confocal immunofluorescence), mitochondrial mass and fragmentation, and mitochondrial bioenergetic profiling were determined. Hypoxia or knockdown of either PPARγ or PGC1α increased HPASMC proliferation, enhanced mitochondria-derived H2O2, decreased mitochondrial mass, stimulated mitochondrial fragmentation, and impaired mitochondrial bioenergetics. Taken together, these findings provide novel evidence that loss of PPARγ diminishes PGC1α and stimulates derangements in mitochondrial structure and function that cause PASMC proliferation. Overexpression of PGC1α reversed hypoxia-induced HPASMC derangements. This study identifies additional mechanistic underpinnings of PH, and provides support for the notion of activating PPARγ as a novel therapeutic strategy in PH.
Sujet(s)
Hypertension pulmonaire/métabolisme , Mitochondries du muscle/métabolisme , Muscles lisses vasculaires/métabolisme , Myocytes du muscle lisse/métabolisme , Récepteur PPAR gamma/métabolisme , Anilides/pharmacologie , Animaux , Hypoxie cellulaire , Prolifération cellulaire , Cellules cultivées , Humains , Hypertension pulmonaire/génétique , Hypertension pulmonaire/anatomopathologie , Hypertension pulmonaire/prévention et contrôle , Souris de lignée C57BL , Mitochondries du muscle/effets des médicaments et des substances chimiques , Mitochondries du muscle/anatomopathologie , Muscles lisses vasculaires/effets des médicaments et des substances chimiques , Muscles lisses vasculaires/anatomopathologie , Myocytes du muscle lisse/effets des médicaments et des substances chimiques , Myocytes du muscle lisse/anatomopathologie , Récepteur PPAR gamma/antagonistes et inhibiteurs , Récepteur PPAR gamma/génétique , Coactivateur 1-alpha du récepteur gamma activé par les proliférateurs de peroxysomes/génétique , Coactivateur 1-alpha du récepteur gamma activé par les proliférateurs de peroxysomes/métabolisme , Artère pulmonaire/effets des médicaments et des substances chimiques , Artère pulmonaire/métabolisme , Artère pulmonaire/anatomopathologie , Interférence par ARNRÉSUMÉ
Pulmonary hypertension (PH) is characterized by increased pulmonary vascular resistance, pulmonary vascular remodeling, and increased pulmonary vascular pressures that often result in right ventricular dysfunction, leading to right heart failure. Evidence suggests that reactive oxygen species (ROS) contribute to PH pathogenesis by altering pulmonary vascular cell proliferation and intracellular signaling pathways. However, the role of mitochondrial antioxidants and oxidant-derived stress signaling in the development of hypoxia-induced PH is largely unknown. Therefore, we examined the role of the major mitochondrial redox regulator thioredoxin 2 (Trx2). Levels of Trx2 mRNA and protein were examined in human pulmonary arterial endothelial cells (HPAECs) and smooth muscle cells (HPASMCs) exposed to hypoxia, a common stimulus for PH, for 72 h. Hypoxia decreased Trx2 mRNA and protein levels. In vitro overexpression of Trx2 reduced hypoxia-induced H2O2 production. The effects of increased Trx2 protein level were examined in transgenic mice expressing human Trx2 (TghTrx2) that were exposed to hypoxia (10% O2) for 3 wk. TghTrx2 mice exposed to hypoxia had exacerbated increases in right ventricular systolic pressures, right ventricular hypertrophy, and increased ROS in the lung tissue. Trx2 overexpression did not attenuate hypoxia-induced increases in Trx2 oxidation or Nox4 expression. Expression of a dominant negative C93S Trx2 mutant that mimics Trx2 oxidation exacerbated hypoxia-induced increases in HPASMC H2O2 levels and cell proliferation. In conclusion, Trx2 overexpression failed to attenuate hypoxia-induced HPASMC proliferation in vitro or hypoxia-induced PH in vivo. These findings indicate that strategies to enhance Trx2 expression are unlikely to exert therapeutic effects in PH pathogenesis.
Sujet(s)
Hypertension pulmonaire/complications , Hypertension pulmonaire/métabolisme , Hypoxie/complications , Hypoxie/métabolisme , Mitochondries/métabolisme , Thiorédoxines/métabolisme , Animaux , Marqueurs biologiques/métabolisme , Prolifération cellulaire/effets des médicaments et des substances chimiques , Modèles animaux de maladie humaine , Humains , Hypertension pulmonaire/anatomopathologie , Hypoxie/anatomopathologie , Souris de lignée C57BL , Souris transgéniques , Protéines mutantes/métabolisme , Myocytes du muscle lisse/effets des médicaments et des substances chimiques , Myocytes du muscle lisse/métabolisme , Oxydoréduction/effets des médicaments et des substances chimiques , Oxygène/pharmacologie , Espèces réactives de l'oxygène/métabolismeRÉSUMÉ
Manganese (Mn) is an abundant redox-active metal with well-characterized mitochondrial accumulation and neurotoxicity due to excessive exposures. Mn is also an essential co-factor for the mitochondrial antioxidant protein, superoxide dismutase-2 (SOD2), and the range for adequate intake established by the Institute of Medicine Food and Nutrition Board is 20% of the interim guidance value for toxicity by the Agency for Toxic Substances and Disease Registry, leaving little margin for safety. To study toxic mechanisms over this critical dose range, we treated human neuroblastoma SH-SY5Y cells with a series of MnCl2 concentrations (from 0 to 100 µM) and measured cellular content to compare to human brain Mn content. Concentrations ≤10 µM gave cellular concentrations comparable to literature values for normal human brain, whereas concentrations ≥50 µM resulted in values comparable to brains from individuals with toxic Mn exposures. Cellular oxygen consumption rate increased as a function of Mn up to 10 µM and decreased with Mn dose ≥50 µM. Over this range, Mn had no effect on superoxide production as measured by aconitase activity or MitoSOX but increased H2O2 production as measured by MitoPY1. Consistent with increased production of H2O2, SOD2 activity, and steady-state oxidation of total thiol increased with increasing Mn. These findings have important implications for Mn toxicity by re-directing attention from superoxide anion radical to H2O2-dependent mechanisms and to investigation over the entire physiologic range to toxicologic range. Additionally, the results show that controlled Mn exposure provides a useful cell manipulation for toxicological studies of mitochondrial H2O2 signaling.
Sujet(s)
Peroxyde d'hydrogène/métabolisme , Manganèse/toxicité , Mitochondries/effets des médicaments et des substances chimiques , Neuroblastome/anatomopathologie , Encéphale/métabolisme , Lignée cellulaire tumorale , Relation dose-effet des médicaments , Humains , Manganèse/métabolisme , Mitochondries/enzymologie , Mitochondries/métabolisme , Oxydoréduction , Superoxide dismutase/métabolismeRÉSUMÉ
Phospholipase C-ε (PLC-ε) is a unique PLC isoform that can be regulated by multiple signaling inputs from both Ras family GTPases and heterotrimeric G proteins and has primary sites of expression in the heart and lung. Whereas the role of PLC-ε in cardiac function and pathology has been documented, its relevance in acute lung injury (ALI) is unclear. We used PLC-ε(-/-) mice to address the role of PLC-ε in regulating lung vascular inflammation and injury in an aerosolized bacterial LPS inhalation mouse model of ALI. PLC-ε(-/-) mice showed a marked decrease in LPS-induced proinflammatory mediators (ICAM-1, VCAM-1, TNF-α, IL-1ß, IL-6, macrophage inflammatory protein 2, keratinocyte-derived cytokine, monocyte chemoattractant protein 1, and granulocyte-macrophage colony-stimulating factor), lung neutrophil infiltration and microvascular leakage, and loss of VE-cadherin compared with PLC-ε(+/+) mice. These data identify PLC-ε as a critical determinant of proinflammatory and leaky phenotype of the lung. To test the possibility that PLC-ε activity in endothelial cells (EC) could contribute to ALI, we determined its role in EC inflammation and barrier disruption. RNAi knockdown of PLC-ε inhibited NF-κB activity in response to diverse proinflammatory stimuli, thrombin, LPS, TNF-α, and the nonreceptor agonist phorbol 13-myristate 12-acetate (phorbol esters) in EC. Depletion of PLC-ε also inhibited thrombin-induced expression of NF-κB target gene, VCAM-1. Importantly, PLC-ε knockdown also protected against thrombin-induced EC barrier disruption by inhibiting the loss of VE-cadherin at adherens junctions and formation of actin stress fibers. These data identify PLC-ε as a novel regulator of EC inflammation and permeability and show a hitherto unknown role of PLC-ε in the pathogenesis of ALI.
Sujet(s)
Lésion pulmonaire aigüe/enzymologie , Phosphoinositide Phospholipase C/physiologie , Lésion pulmonaire aigüe/immunologie , Lésion pulmonaire aigüe/anatomopathologie , Animaux , Antigènes CD/métabolisme , Cadhérines/métabolisme , Perméabilité capillaire , Cellules cultivées , Endothélium vasculaire/enzymologie , Endothélium vasculaire/anatomopathologie , Humains , Poumon/vascularisation , Poumon/anatomopathologie , Souris de lignée C57BL , Souris knockout , Facteur de transcription NF-kappa B/métabolisme , Transduction du signal , Fibres de stress/métabolisme , Vascularite/enzymologieRÉSUMÉ
BACKGROUND: Pathogenesis of pulmonary hypertension is complex and involves activation of the transcription factor, hypoxia-inducible factor-1 (HIF-1) that shifts cellular metabolism from aerobic respiration to glycolysis, in part, by increasing the expression of its downstream target pyruvate dehydrogenase kinase-1 (PDK-1), thereby promoting a proliferative, apoptosis-resistant phenotype in pulmonary vascular cells. Activation of the nuclear hormone transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), attenuates pulmonary hypertension and pulmonary artery smooth muscle cell (PASMC) proliferation. In the current study, we determined whether PPARγ inhibits HIF-1α and PDK-1 expression in human PASMCs. METHODS: HPASMCs were exposed to normoxia (21% O2) or hypoxia (1% O2) for 2-72 hours ± treatment with the PPARγ-ligand, rosiglitazone (RSG, 10µM). RESULTS: Compared to normoxia, HIF-1α mRNA levels were elevated in HPASMC at 2 hours hypoxia and reduced to baseline levels by 24-72 hours. HIF-1α protein levels increased following 4 and 8 hours of hypoxia and returned to baseline levels by 24 and 72 hours. PDK-1 protein levels increased following 24 hours hypoxia and remained elevated by 72 hours. RSG treatment at the onset of hypoxia attenuated HIF-1α protein and PDK-1 mRNA and protein levels at 4, 8 and 24 hours of hypoxia, respectively. However, RSG treatment during final 24 hours of 72-hour hypoxia, an intervention that inhibits HPASMC proliferation, failed to prevent hypoxia-induced PDK-1 expression. CONCLUSION: Hypoxia causes transient activation of HPASMC HIF-1α that is attenuated by RSG treatment initiated at hypoxia onset. These findings provide novel evidence that PPARγ modulates fundamental and acute cellular responses to hypoxia through both HIF-1-dependent and HIF-1-independent mechanisms.
Sujet(s)
Sous-unité alpha du facteur-1 induit par l'hypoxie/génétique , Récepteur PPAR gamma/génétique , Transduction du signal , Thiazolidinediones/pharmacologie , Prolifération cellulaire/effets des médicaments et des substances chimiques , Humains , Hypoxie/physiopathologie , Sous-unité alpha du facteur-1 induit par l'hypoxie/métabolisme , Ligands , Myocytes du muscle lisse/physiologie , Récepteur PPAR gamma/métabolisme , Protein-Serine-Threonine Kinases/génétique , Protein-Serine-Threonine Kinases/métabolisme , Artère pulmonaire/physiopathologie , Pyruvate dehydrogenase acetyl-transferring kinase , RosiglitazoneRÉSUMÉ
Hypoxia stimulates pulmonary hypertension (PH), in part by increasing the proliferation of human pulmonary artery smooth muscle cells (HPASMCs) via sustained activation of mitogen-activated protein kinase, extracellular signal-regulated kinases 1 and 2 (ERK 1/2), and nuclear factor-kappa B (NF-κB); elevated expression of NADPH oxidase 4 (Nox4); and downregulation of peroxisome proliferator-activated receptor gamma (PPARγ) levels. However, the upstream mediators that control these responses remain largely unknown. We hypothesized that proline-rich tyrosine kinase 2 (Pyk2) plays a critical role in the mechanism of hypoxia-induced HPASMC proliferation. To test this hypothesis, HPASMCs were exposed to normoxia or hypoxia (1% O2) for 72 hours. Hypoxia activated Pyk2 (detected as Tyr402 phosphorylation), and inhibition of Pyk2 with small interfering RNA (siRNA) or tyrphostin A9 attenuated hypoxia-induced HPASMC proliferation. Pyk2 inhibition attenuated ERK 1/2 activation as early as 24 hours after the onset of hypoxia, suggesting a proximal role for Pyk2 in this response. Pyk2 inhibition also attenuated hypoxia-induced NF-κB activation, reduced HPASMC PPARγ messenger RNA levels and activity, and increased NF-κB-mediated Nox4 levels. The siRNA-mediated PPARγ knockdown enhanced Pyk2 activation, whereas PPARγ overexpression reduced Pyk2 activation in HPASMCs, confirming a reciprocal relationship between Pyk2 and PPARγ. Pyk2 depletion also attenuated hypoxia-induced NF-κB p65 activation and reduced PPARγ protein levels in human pulmonary artery endothelial cells. These in vitro findings suggest that Pyk2 plays a central role in the proliferative phenotype of pulmonary vascular wall cells under hypoxic conditions. Coupled with recent reports that hypoxia-induced PH is attenuated in Pyk2 knockout mice, these findings suggest that Pyk2 may represent a novel therapeutic target in PH.
RÉSUMÉ
Serine proteinase inhibitor, clade E, member 2 (SERPINE2), is a cell- and extracellular matrix-associated inhibitor of thrombin. Although SERPINE2 is a candidate susceptibility gene for chronic obstructive pulmonary disease, the physiologic role of this protease inhibitor in lung development and homeostasis is unknown. We observed spontaneous monocytic-cell infiltration in the lungs of Serpine2-deficient (SE2(-/-)) mice, beginning at or before the time of lung maturity, which resulted in lesions that resembled bronchus-associated lymphoid tissue (BALT). The initiation of lymphocyte accumulation in the lungs of SE2(-/-) mice involved the excessive expression of chemokines, cytokines, and adhesion molecules that are essential for BALT induction, organization, and maintenance. BALT-like lesion formation in the lungs of SE2(-/-) mice was also associated with a significant increase in the activation of thrombin, a recognized target of SE2, and excess stimulation of NF-κB, a major regulator of chemokine expression and inflammation. Finally, systemic delivery of thrombin rapidly stimulated lung chemokine expression in vivo These data uncover a novel mechanism whereby loss of serine protease inhibition leads to lung lymphocyte accumulation.-Solleti, S. K., Srisuma, S., Bhattacharya, S., Rangel-Moreno, J., Bijli, K. M., Randall, T. D., Rahman, A., Mariani, T. J. Serpine2 deficiency results in lung lymphocyte accumulation and bronchus-associated lymphoid tissue formation.
Sujet(s)
Bronches/anatomopathologie , Poumon/cytologie , Lymphocytes/physiologie , Tissu lymphoïde/anatomopathologie , Serpine E2/métabolisme , Animaux , Régulation de l'expression des gènes/physiologie , Souris , Souris knockout , Serpine E2/génétiqueRÉSUMÉ
Endothelin-1 (ET-1) plays a critical role in endothelial dysfunction and contributes to the pathogenesis of pulmonary hypertension (PH). We hypothesized that peroxisome proliferator-activated receptor γ (PPARγ) stimulates microRNAs that inhibit ET-1 and pulmonary artery endothelial cell (PAEC) proliferation. The objective of this study was to clarify molecular mechanisms by which PPARγ regulates ET-1 expression in vitro and in vivo. In PAECs isolated from patients with pulmonary arterial hypertension, microRNA (miR)-98 expression was reduced, and ET-1 protein levels and proliferation were increased. Similarly, hypoxia reduced miR-98 and increased ET-1 levels and PAEC proliferation in vitro. In vivo, hypoxia reduced miR-98 expression and increased ET-1 and proliferating cell nuclear antigen (PCNA) levels in mouse lung, derangements that were aggravated by treatment with the vascular endothelial growth factor receptor antagonist Sugen5416. Reporter assays confirmed that miR-98 binds directly to the ET-1 3'-untranslated region. Compared with littermate control mice, miR-98 levels were reduced and ET-1 and PCNA expression were increased in lungs from endothelial-targeted PPARγ knockout mice, whereas miR-98 levels were increased and ET-1 and PCNA expression was reduced in lungs from endothelial-targeted PPARγ-overexpression mice. Gain or loss of PPARγ function in PAECs in vitro confirmed that alterations in PPARγ were sufficient to regulate miR-98, ET-1, and PCNA expression. Finally, PPARγ activation with rosiglitazone regimens that attenuated hypoxia-induced PH in vivo and human PAEC proliferation in vitro restored miR-98 levels. The results of this study show that PPARγ regulates miR-98 to modulate ET-1 expression and PAEC proliferation. These results further clarify molecular mechanisms by which PPARγ participates in PH pathogenesis and therapy.
Sujet(s)
Cellules endothéliales/métabolisme , Endothéline-1/métabolisme , Hypertension pulmonaire/métabolisme , Hypoxie/métabolisme , microARN/métabolisme , Récepteur PPAR gamma/métabolisme , Artère pulmonaire/métabolisme , Transduction du signal , Régions 3' non traduites , Animaux , Sites de fixation , Prolifération cellulaire , Cellules cultivées , Cellules endothéliales/effets des médicaments et des substances chimiques , Cellules endothéliales/anatomopathologie , Endothéline-1/génétique , Régulation de l'expression des gènes , Humains , Hypertension pulmonaire/induit chimiquement , Hypertension pulmonaire/traitement médicamenteux , Hypertension pulmonaire/génétique , Hypertension pulmonaire/anatomopathologie , Hypoxie/complications , Hypoxie/génétique , Hypoxie/anatomopathologie , Indoles , Mâle , Souris de lignée C57BL , Souris knockout , microARN/génétique , Récepteur PPAR gamma/agonistes , Récepteur PPAR gamma/déficit , Récepteur PPAR gamma/génétique , Artère pulmonaire/effets des médicaments et des substances chimiques , Artère pulmonaire/anatomopathologie , Pyrroles , Interférence par ARN , Rosiglitazone , Transduction du signal/effets des médicaments et des substances chimiques , Thiazolidinediones/pharmacologie , Transfection , Remodelage vasculaireRÉSUMÉ
Pulmonary hypertension (PH) is characterized by increased pulmonary vascular remodeling, resistance, and pressures. Reactive oxygen species (ROS) contribute to PH-associated vascular dysfunction. NADPH oxidases (Nox) and mitochondria are major sources of superoxide (O(2)(â¢-)) and hydrogen peroxide (H(2)O(2)) in pulmonary vascular cells. Hypoxia, a common stimulus of PH, increases Nox expression and mitochondrial ROS (mtROS) production. The interactions between these two sources of ROS generation continue to be defined. We hypothesized that mitochondria-derived O(2)(â¢-) (mtO(2)(â¢-)) and H(2)O(2) (mtH(2)O(2)) increase Nox expression to promote PH pathogenesis and that mitochondria-targeted antioxidants can reduce mtROS, Nox expression, and hypoxia-induced PH. Exposure of human pulmonary artery endothelial cells to hypoxia for 72 h increased mtO(2)(â¢-) and mtH(2)O(2). To assess the contribution of mtO(2)(â¢-) and mtH(2)O(2) to hypoxia-induced PH, mice that overexpress superoxide dismutase 2 (Tg(hSOD2)) or mitochondria-targeted catalase (MCAT) were exposed to normoxia (21% O(2)) or hypoxia (10% O(2)) for three weeks. Compared with hypoxic control mice, MCAT mice developed smaller hypoxia-induced increases in RVSP, α-SMA staining, extracellular H(2)O(2) (Amplex Red), Nox2 and Nox4 (qRT-PCR and Western blot), or cyclinD1 and PCNA (Western blot). In contrast, Tg(hSOD2) mice experienced exacerbated responses to hypoxia. These studies demonstrate that hypoxia increases mtO(2)(â¢-) and mtH(2)O(2). Targeting mtH(2)O(2) attenuates PH pathogenesis, whereas targeting mtO(2)(â¢-) exacerbates PH. These differences in PH pathogenesis were mirrored by RVSP, vessel muscularization, levels of Nox2 and Nox4, proliferation, and H(2)O(2) release. These studies suggest that targeted reductions in mtH(2)O(2) generation may be particularly effective in preventing hypoxia-induced PH.
Sujet(s)
Hypoxie cellulaire , Cellules endothéliales/métabolisme , Hypertension pulmonaire/métabolisme , Mitochondries/métabolisme , Espèces réactives de l'oxygène/métabolisme , Animaux , Antioxydants/métabolisme , Antioxydants/pharmacologie , Prolifération cellulaire , Cellules endothéliales/anatomopathologie , Humains , Hypertension pulmonaire/anatomopathologie , Glycoprotéines membranaires/métabolisme , Souris , Mitochondries/anatomopathologie , NADPH Oxidase 2 , NADPH Oxidase 4 , NADPH oxidase/métabolisme , Phénytoïne/analogues et dérivés , Phénytoïne/métabolisme , Artère pulmonaire/métabolisme , Artère pulmonaire/anatomopathologie , Superoxide dismutase/métabolismeRÉSUMÉ
Chronic obstructive pulmonary disease (COPD) is a highly prevalent, chronic inflammatory lung disease with limited existing therapeutic options. While modulation of peroxisome proliferator-activating receptor (PPAR)-γ activity can modify inflammatory responses in several models of lung injury, the relevance of the PPARG pathway in COPD pathogenesis has not been previously explored. Mice lacking Pparg specifically in airway epithelial cells displayed increased susceptibility to chronic cigarette smoke (CS)-induced emphysema, with excessive macrophage accumulation associated with increased expression of chemokines, Ccl5, Cxcl10, and Cxcl15. Conversely, treatment of mice with a pharmacological PPARγ activator attenuated Cxcl10 and Cxcl15 expression and macrophage accumulation in response to CS. In vitro, CS increased lung epithelial cell chemokine expression in a PPARγ activation-dependent fashion. The ability of PPARγ to regulate CS-induced chemokine expression in vitro was not specifically associated with peroxisome proliferator response element (PPRE)-mediated transactivation activity but was correlated with PPARγ-mediated transrepression of NF-κB activity. Pharmacological or genetic activation of PPARγ activity abrogated CS-dependent induction of NF-κB activity. Regulation of NF-κB activity involved direct PPARγ-NF-κB interaction and PPARγ-mediated effects on IKK activation, IκBα degradation, and nuclear translocation of p65. Our data indicate that PPARG represents a disease-relevant pathophysiological and pharmacological target in COPD. Its activation state likely contributes to NF-κB-dependent, CS-induced chemokine-mediated regulation of inflammatory cell accumulation.
Sujet(s)
Chimiokines/métabolisme , Récepteur PPAR gamma/physiologie , Emphysème pulmonaire/métabolisme , Fumer/effets indésirables , Animaux , Lignée cellulaire , Prédisposition aux maladies , Femelle , Souris de souche-129 , Souris de lignée C57BL , Facteur de transcription NF-kappa B/métabolisme , Emphysème pulmonaire/étiologie , Emphysème pulmonaire/immunologie , Transduction du signal , Fumer/immunologie , Fumer/métabolisme , Activation de la transcriptionRÉSUMÉ
Hypoxia stimulates pulmonary hypertension (PH) in part by increasing the proliferation of pulmonary vascular wall cells. Recent evidence suggests that signaling events involved in hypoxia-induced cell proliferation include sustained nuclear factor-kappaB (NF-κB) activation, increased NADPH oxidase 4 (Nox4) expression, and downregulation of peroxisome proliferator-activated receptor gamma (PPARγ) levels. To further understand the role of reduced PPARγ levels associated with PH pathobiology, siRNA was employed to reduce PPARγ levels in human pulmonary artery smooth muscle cells (HPASMC) in vitro under normoxic conditions. PPARγ protein levels were reduced to levels comparable to those observed under hypoxic conditions. Depletion of PPARγ for 24-72 h activated mitogen-activated protein kinase, ERK 1/2, and NF-κB. Inhibition of ERK 1/2 prevented NF-κB activation caused by PPARγ depletion, indicating that ERK 1/2 lies upstream of NF-κB activation. Depletion of PPARγ for 72 h increased NF-κB-dependent Nox4 expression and H2O2 production. Inhibition of NF-κB or Nox4 attenuated PPARγ depletion-induced HPASMC proliferation. Degradation of PPARγ depletion-induced H2O2 by PEG-catalase prevented HPASMC proliferation and also ERK 1/2 and NF-κB activation and Nox4 expression, indicating that H2O2 participates in feed-forward activation of the above signaling events. Contrary to the effects of PPARγ depletion, HPASMC PPARγ overexpression reduced ERK 1/2 and NF-κB activation, Nox4 expression, and cell proliferation. Taken together these findings provide novel evidence that PPARγ plays a central role in the regulation of the ERK1/2-NF-κB-Nox4-H2O2 signaling axis in HPASMC. These results indicate that reductions in PPARγ caused by pathophysiological stimuli such as prolonged hypoxia exposure are sufficient to promote the proliferation of pulmonary vascular smooth muscle cells observed in PH pathobiology.
Sujet(s)
Cellules endothéliales/enzymologie , NADPH oxidase/génétique , Facteur de transcription NF-kappa B/génétique , Récepteur PPAR gamma/génétique , Anticorps neutralisants/pharmacologie , Catalase/pharmacologie , Hypoxie cellulaire , Prolifération cellulaire/effets des médicaments et des substances chimiques , Cellules cultivées , Cellules endothéliales/cytologie , Cellules endothéliales/effets des médicaments et des substances chimiques , Flavonoïdes/pharmacologie , Régulation de l'expression des gènes , Humains , Peroxyde d'hydrogène/métabolisme , Mitogen-Activated Protein Kinase 1/antagonistes et inhibiteurs , Mitogen-Activated Protein Kinase 1/génétique , Mitogen-Activated Protein Kinase 1/métabolisme , Mitogen-Activated Protein Kinase 3/antagonistes et inhibiteurs , Mitogen-Activated Protein Kinase 3/génétique , Mitogen-Activated Protein Kinase 3/métabolisme , Modèles biologiques , NADPH Oxidase 4 , NADPH oxidase/métabolisme , Facteur de transcription NF-kappa B/métabolisme , Récepteur PPAR gamma/antagonistes et inhibiteurs , Récepteur PPAR gamma/métabolisme , Polyéthylène glycols/pharmacologie , Inhibiteurs de protéines kinases/pharmacologie , Petit ARN interférent/génétique , Petit ARN interférent/métabolisme , Transduction du signalRÉSUMÉ
RATIONALE: Airway hyperresponsiveness (AHR) is classically found in asthma, and persistent AHR is associated with poor asthma control. Although airway smooth muscle (ASM) cells play a critical pathophysiologic role in AHR, the paracrine contributions of surrounding cells such as fibroblasts to the contractile phenotype of ASM cells have not been examined fully. This study addresses the hypothesis that nicotine promotes a contractile ASM cell phenotype by stimulating fibroblasts to increase nerve growth factor (NGF) secretion into the environment. METHODS: Primary lung fibroblasts isolated from wild type and α7 nicotinic acetylcholine receptor (α7 nAChR) deficient mice were treated with nicotine (50 µg/ml) in vitro for 72 hours. NGF levels were measured in culture media and in bronchoalveolar lavage (BAL) fluid from asthmatic, smoking and non-smoking subjects by ELISA. The role of the NFκB pathway in nicotine-induced NGF expression was investigated by measuring NFκB nuclear translocation, transcriptional activity, chromatin immunoprecipitation assays, and si-p65 NFκB knockdown. The ability of nicotine to stimulate a fibroblast-mediated, contractile ASM cell phenotype was confirmed by examining expression of contractile proteins in ASM cells cultured with fibroblast-conditioned media or BAL fluid. RESULTS: NGF levels were elevated in the bronchoalveolar lavage fluid of nicotine-exposed mice, current smokers, and asthmatic children. Nicotine increased NGF secretion in lung fibroblasts in vitro in a dose-dependent manner and stimulated NFκB nuclear translocation, p65 binding to the NGF promoter, and NFκB transcriptional activity. These responses were attenuated in α7 nAChR deficient fibroblasts and in wild type fibroblasts following NFκB inhibition. Nicotine-treated, fibroblast-conditioned media increased expression of contractile proteins in ASM cells. CONCLUSION: Nicotine stimulates NGF release by lung fibroblasts through α7 nAChR and NFκB dependent pathways. These novel findings suggest that the nicotine-α7 nAChR-NFκB- NGF axis may provide novel therapeutic targets to attenuate tobacco smoke-induced AHR.
Sujet(s)
Fibroblastes/effets des médicaments et des substances chimiques , Fibroblastes/métabolisme , Poumon/cytologie , Facteur de transcription NF-kappa B/métabolisme , Facteur de croissance nerveuse/métabolisme , Nicotine/pharmacologie , Animaux , Régulation de l'expression des gènes/effets des médicaments et des substances chimiques , Humains , Souris , Récepteur nicotinique de l'acétylcholine alpha7/métabolismeRÉSUMÉ
We addressed the role of transglutaminase 2 (TG2), a calcium-dependent enzyme that catalyzes cross-linking of proteins, in the mechanism of endothelial cell (EC) inflammation and lung polymorphonuclear lymphocyte (PMN) infiltration. Exposure of EC to thrombin, a procoagulant and proinflammatory mediator, resulted in activation of the transcription factor nuclear factor κB (NF-κB) and its target genes, vascular cell adhesion molecule 1, monocyte chemotactic protein 1, and interleukin 6. RNAi knockdown of TG2 inhibited these responses. Analysis of NF-κB activation pathway showed that TG2 knockdown was associated with inhibition of thrombin-induced DNA binding as well as serine phosphorylation of RelA/p65, a crucial event that controls transcriptional capacity of the DNA-bound RelA/p65. These results implicate an important role for TG2 in mediating EC inflammation by promoting DNA-binding and transcriptional activity of RelA/p65. Because thrombin is released in high amounts during sepsis, and its concentration is elevated in plasma and lavage fluids of patients with acute respiratory distress syndrome, we determined the in vivo relevance of TG2 in a mouse model of sepsis-induced lung PMN recruitment. A marked reduction in NF-κB activation, adhesion molecule expression, and lung PMN sequestration was observed in TG2 knockout mice compared with wild-type mice exposed to endotoxemia. Together, these results identify TG2 as an important mediator of EC inflammation and lung PMN sequestration associated with intravascular coagulation and sepsis.
Sujet(s)
Protéines G/métabolisme , Régulation de l'expression des gènes codant pour des enzymes , Inflammation/métabolisme , Granulocytes neutrophiles/cytologie , Transglutaminases/métabolisme , Animaux , Adhérence cellulaire , Cellules cultivées , Cellules endothéliales/enzymologie , Endotoxémie/métabolisme , Fibrose/physiopathologie , Humains , Poumon/métabolisme , Lymphocytes/cytologie , Souris , Souris de lignée C57BL , Souris knockout , Facteur de transcription NF-kappa B/métabolisme , Phosphorylation , Protein glutamine gamma glutamyltransferase-2 , Artère pulmonaire/métabolisme , Thrombine/métabolisme , Facteur de transcription RelA/métabolismeRÉSUMÉ
Pulmonary hypertension (PH) is a serious disorder that causes significant morbidity and mortality. The pathogenesis of PH involves complex derangements in multiple pathways including reductions in peroxisome proliferator-activated receptor gamma (PPARγ). Hypoxia, a common PH stimulus, reduces PPARγ in experimental models. In contrast, activating PPARγ attenuates hypoxia-induced PH and endothelin 1 (ET-1) expression. To further explore mechanisms of hypoxia-induced PH and reductions in PPARγ, we examined the effects of hypoxia on selected microRNA (miRNA or miR) levels that might reduce PPARγ expression leading to increased ET-1 expression and PH. Our results demonstrate that exposure to hypoxia (10% O2) for 3-weeks increased levels of miR-27a and ET-1 in the lungs of C57BL/6 mice and reduced PPARγ levels. Hypoxia-induced increases in miR-27a were attenuated in mice treated with the PPARγ ligand, rosiglitazone (RSG, 10 mg/kg/d) by gavage for the final 10 d of exposure. In parallel studies, human pulmonary artery endothelial cells (HPAECs) were exposed to control (21% O2) or hypoxic (1% O2) conditions for 72 h. Hypoxia increased HPAEC proliferation, miR-27a and ET-1 expression, and reduced PPARγ expression. These alterations were attenuated by treatment with RSG (10 µM) during the last 24 h of hypoxia exposure. Overexpression of miR-27a or PPARγ knockdown increased HPAEC proliferation and ET-1 expression and decreased PPARγ levels, whereas these effects were reversed by miR-27a inhibition. Further, compared to lungs from littermate control mice, miR-27a levels were upregulated in lungs from endothelial-targeted PPARγ knockout (ePPARγ KO) mice. Knockdown of either SP1 or EGR1 was sufficient to significantly attenuate miR-27a expression in HPAECs. Collectively, these studies provide novel evidence that miR-27a and PPARγ mediate mutually repressive actions in hypoxic pulmonary vasculature and that targeting PPARγ may represent a novel therapeutic approach in PH to attenuate proliferative mediators that stimulate proliferation of pulmonary vascular cells.
Sujet(s)
Hypoxie/métabolisme , Poumon/vascularisation , Poumon/métabolisme , microARN/génétique , Récepteur PPAR gamma/métabolisme , Animaux , Appariement de bases , Séquence nucléotidique , Prolifération cellulaire , Modèles animaux de maladie humaine , Facteur de transcription EGR-1/génétique , Cellules endothéliales/métabolisme , Endothéline-1/métabolisme , Expression des gènes , Régulation de l'expression des gènes , Techniques de knock-down de gènes , Humains , Hypertension pulmonaire/génétique , Hypertension pulmonaire/métabolisme , Souris , microARN/composition chimique , microARN/métabolisme , Modèles biologiques , Récepteur PPAR gamma/composition chimique , Récepteur PPAR gamma/génétique , Artère pulmonaire/métabolisme , Interférence par ARN , Facteur de transcription Sp1/génétiqueRÉSUMÉ
The ligand-activated transcription factor peroxisome proliferator-activated receptor γ (PPARγ) regulates metabolism, cell proliferation, and inflammation. Pulmonary hypertension (PH) is associated with reduced PPARγ expression, and hypoxia exposure regimens that cause PH reduce PPARγ expression. This study examines mechanisms of hypoxia-induced PPARγ downregulation in vitro and in vivo. Hypoxia reduced PPARγ mRNA and protein levels, PPARγ activity, and the expression of PPARγ-regulated genes in human pulmonary artery smooth muscle cells (HPASMCs) exposed to 1% oxygen for 72 h. Similarly, exposure of mice to hypoxia (10% O2) for 3 weeks reduced PPARγ mRNA and protein in mouse lung. Inhibiting ERK1/2 with PD98059 or treatment with siRNA directed against either NF-κB p65 or Nox4 attenuated hypoxic reductions in PPARγ expression and activity. Furthermore, degradation of H2O2 using PEG-catalase prevented hypoxia-induced ERK1/2 phosphorylation and Nox4 expression, suggesting sustained ERK1/2-mediated signaling and Nox4 expression in this response. Mammalian two-hybrid assays demonstrated that PPARγ and p65 bind directly to each other in a mutually repressive fashion. We conclude from these results that hypoxic regimens that promote PH pathogenesis and HPASMC proliferation reduce PPARγ expression and activity through ERK1/2-, p65-, and Nox4-dependent pathways. These findings provide novel insights into mechanisms by which pathophysiological stimuli such as hypoxia cause loss of PPARγ activity and pulmonary vascular cell proliferation, pulmonary vascular remodeling, and PH. These results also indicate that restoration of PPARγ activity with pharmacological ligands may provide a novel therapeutic approach in selected forms of PH.
Sujet(s)
Hypertension pulmonaire/métabolisme , Myocytes du muscle lisse/métabolisme , Récepteur PPAR gamma/métabolisme , Artère pulmonaire/métabolisme , Animaux , Catalase/métabolisme , Lignée cellulaire , Prolifération cellulaire/effets des médicaments et des substances chimiques , Humains , Peroxyde d'hydrogène/métabolisme , Peroxyde d'hydrogène/pharmacologie , Hypertension pulmonaire/génétique , Hypertension pulmonaire/anatomopathologie , Hypoxie/métabolisme , Hypoxie/anatomopathologie , Système de signalisation des MAP kinases/génétique , Souris , Myocytes du muscle lisse/cytologie , NADPH Oxidase 4 , NADPH oxidase/génétique , NADPH oxidase/métabolisme , Facteur de transcription NF-kappa B/métabolisme , Récepteur PPAR gamma/génétique , Polyéthylène glycols/métabolisme , Artère pulmonaire/cytologie , Artère pulmonaire/anatomopathologie , Petit ARN interférent , Transduction du signalRÉSUMÉ
The pathogenesis of acute lung injury (ALI) involves bidirectional cooperation and close interaction between inflammatory and coagulation pathways. A key molecule linking coagulation and inflammation is the procoagulant thrombin, a serine protease whose concentration is elevated in plasma and lavage fluids of patients with ALI and acute respiratory distress syndrome (ARDS). However, little is known about the mechanism by which thrombin contributes to lung inflammatory response. In this study, we developed a new mouse model that permits investigation of lung inflammation associated with intravascular coagulation. Using this mouse model and in vitro approaches, we addressed the role of non-muscle myosin light chain kinase (nmMLCK) in thrombin-induced endothelial cell (EC) inflammation and lung neutrophil (PMN) infiltration. Our in vitro experiments revealed a key role of nmMLCK in ICAM-1 expression by its ability to control nuclear translocation and transcriptional capacity of RelA/p65 in EC. When subjected to intraperitoneal thrombin challenge, wild type mice showed a marked increase in lung PMN infiltration via expression of ICAM-1. However, these responses were markedly attenuated in mice deficient in nmMLCK. These results provide mechanistic insight into lung inflammatory response associated with intravascular coagulation and identify nmMLCK as a critical target for modulation of lung inflammation.
Sujet(s)
Cellules endothéliales/cytologie , Régulation de l'expression des gènes , Agranulocytes/cytologie , Poumon/métabolisme , Myosin-Light-Chain Kinase/physiologie , Thrombine/métabolisme , Animaux , Coagulation sanguine , Noyau de la cellule/métabolisme , Cellules endothéliales/métabolisme , Femelle , Cellules endothéliales de la veine ombilicale humaine , Humains , Inflammation , Molécule-1 d'adhérence intercellulaire/métabolisme , Mâle , Souris , Souris de lignée C57BL , Souris transgéniques , Granulocytes neutrophiles/métabolisme , Myeloperoxidase/métabolisme , Facteur de transcription RelA/métabolismeRÉSUMÉ
We investigated the role of proline-rich tyrosine kinase 2 (Pyk2) in the mechanism of NF-κB activation and endothelial cell (EC) inflammation induced by thrombin, a procoagulant serine protease released in high amounts during sepsis and other inflammatory conditions. Stimulation of ECs with thrombin resulted in a time-dependent activation of Pyk2. RNA interference knockdown of Pyk2 attenuated thrombin-induced activity of NF-κB and expression of its target genes, vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1. Pyk2 knockdown impaired thrombin-induced activation of IκB kinase (IKK) and phosphorylation (Ser32 and Ser36) of IkappaBα, but, surprisingly, failed to prevent IκBα degradation. However, depletion of IKKα or IKKß was effective in inhibiting IκBα phosphorylation/degradation, as expected. Intriguingly, Pyk2 knockdown impaired nuclear translocation and DNA binding of RelA/p65, despite the inability to prevent IκBα degradation. In addition, Pyk2 knockdown was associated with inhibition of RelA/p65 phosphorylation at Ser536, which is important for transcriptional activity of RelA/p65. Depletion of IKKα or IKKß each impaired RelA/p65 phosphorylation. Taken together, these data identify Pyk2 as a critical regulator of EC inflammation by virtue of engaging IKK to promote the release and the transcriptional capacity of RelA/p65, and, additionally, by its ability to facilitate the nuclear translocation of the released RelA/p65. Thus, specific targeting of Pyk2 may be an effective anti-inflammatory strategy in vascular diseases associated with EC inflammation and intravascular coagulation.