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ABSTRACT: The lung hosts multiple populations of macrophages and dendritic cells, which play a crucial role in lung pathology. Accurate identification and enumeration of these subsets is essential for understanding their role in lung pathology. Flow cytometry is a mainstream tool for studying immune system, however, a systematic flow cytometric approach to accurately and consistently identify subsets of macrophages and dendritic cells (DCs) in the normal mouse lung has not been described. Here we developed a panel of surface markers and an analysis strategy that accurately identifies all known populations of macrophages and DCs, and their precursors in the lung during steady state conditions and bleomycin-induced injury. Using this panel we assessed polarization of lung macrophages during the course of bleomycin-induced lung injury. Alveolar macrophages expressed markers of alternatively activated macrophages during both acute and fibrotic phases of bleomycin-induced lung injury, while markers of classically activated macrophages were expressed only during the acute phase. Taken together, these data suggest that this flow cytometric panel is very helpful for identifying macrophage and DC populations and their state of activation in normal, injured and fibrotic lung.
American Journal of Respiratory Cell and Molecular Biology 05/2013; · 5.13 Impact Factor
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American Journal of Respiratory and Critical Care Medicine 05/2013; 187(9):898-900. · 11.08 Impact Factor
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ABSTRACT: The repair of the bronchiolar epithelium damaged by cell-mediated, physical or chemical insult requires epithelial cell migration over a provisional matrix composed of complexes of extracellular matrix molecules, including fibronectin and laminin. These matrix molecules support migration and enhance cell adhesion. Too much adhesion and cells fail to move, whereas too little adhesion and cells are unable to develop the traction force necessary for motility. Thus, we investigated the relative contributions of laminin and fibronectin to bronchiolar cell adhesion and migration using the immortalized bronchial lung epithelial cell line (BEP2D) and normal human bronchial epithelial (NHBE) cells, both of which assemble a laminin-322/fibronectin-rich matrix. Intriguingly, BEP2D and NHBE cells migrate significantly faster on a laminin-332-rich matrix than on fibronectin. Moreover, addition of fibronectin to laminin-332 matrix suppresses motility of both cell types. Finally, fibronectin enhances the adhesion of both BEP2D and NHBE cells to laminin-332 coated surfaces. These results suggest that fibronectin fine tunes laminin-332-mediated migration by boosting bronchiolar cell adhesion to substrate. We suggest that during epithelial wound healing of the injured airway, fibronectin plays an important adhesive role for laminin-driven epithelial cell motility by promoting a stable cellular interaction with the provisional matrix.
American Journal of Respiratory Cell and Molecular Biology 04/2013; · 5.13 Impact Factor
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ABSTRACT: Oxygen therapy is an integral part of the treatment of critically ill patients. Maintenance of adequate oxygen delivery to vital organs often requires the administration of supplemental oxygen, sometimes at high concentrations. Although oxygen therapy is lifesaving, it may be associated with deleterious effects when administered for prolonged periods at high concentrations. Here, we review the recent advances in our understanding of the molecular responses to hypoxia and high levels of oxygen and review the current guidelines for oxygen therapy in critically ill patients.
Chest 04/2013; 143(4):1151-62. · 5.25 Impact Factor
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Hanh Chi Do-Umehara,
Cong Chen,
Daniela Urich,
Liang Zhou,
Ju Qiu,
Samuel Jang,
Alia Zander,
Margaret A Baker,
Martin Eilers,
Peter H S Sporn,
Karen M Ridge,
Jacob I Sznajder, G R Scott Budinger,
Gökhan M Mutlu,
Anning Lin,
Jing Liu
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ABSTRACT: Inflammation is essential for host defense but can cause tissue damage and organ failure if unchecked. How the inflammation is resolved remains elusive. Here we report that the transcription factor Miz1 was required for terminating lipopolysaccharide (LPS)-induced inflammation. Genetic disruption of the Miz1 POZ domain, which is essential for the transactivation or repression activity of Miz1, resulted in hyperinflammation, lung injury and greater mortality in LPS-treated mice but a lower bacterial load and mortality in mice with Pseudomonas aeruginosa pneumonia. Loss of the Miz1 POZ domain prolonged the expression of proinflammatory cytokines. After stimulation, Miz1 was phosphorylated at Ser178, which was required for recruitment of the histone deacetylase HDAC1 to repress transcription of the gene encoding C/EBP-δ, an amplifier of inflammation. Our data provide a long-sought mechanism underlying the resolution of LPS-induced inflammation.
Nature Immunology 03/2013; · 26.01 Impact Factor
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ABSTRACT: Transforming Growth Factor-beta (TGF-beta) signaling is required for normal tissue repair, however, excessive TGF-beta signaling can lead to robust profibrotic gene expression in fibroblasts resulting in tissue fibrosis. TGF-beta binds to cell surface receptors resulting in the phosphorylation of Smad family of transcription factors to initiate gene expression. TGF-beta also initiates Smad-independent pathways to induce maximal gene expression. The interaction of Smad-dependent and independent pathways in regulating TGF-beta induced gene expression is not fully understood. Here we report that mitochondrial reactive oxygen species (ROS) generated at complex III are required for TGF-beta induced gene expression in primary normal human lung fibroblasts. TGF-beta induced ROS could be detected in both the mitochondrial matrix and cytosol. Mitochondrial targeted antioxidants markedly attenuated TGF-beta induced gene expression without affecting Smad phosphorylation or nuclear translocation. Genetically disrupting mitochondrial complex III generated ROS production attenuated TGF-beta induced profibrotic gene expression. Furthermore, diminishing mitochondrial ROS attenuated NADPH oxidase 4 (NOX4) expression, which is required for TGF-beta induced myofibrobast differentiation. Lung fibroblasts from patients with pulmonary fibrosis generated more mitochondrial ROS than normal human lung fibroblasts, however, mitochondrial targeted antioxidants attenuated profibrotic gene expression in both normal and fibrotic lung fibroblasts. Collectively, our results indicate that mitochondrial ROS are essential for the normal TGF-beta -mediated gene expression and that targeting mitochondrial ROS might be beneficial in diseases associated with excessive fibrosis.
Journal of Biological Chemistry 11/2012; · 4.77 Impact Factor
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ABSTRACT: Alcohol intake increases the risk of acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS) and is associated with poor outcomes in patients who develop these syndromes. No specific therapies are currently available to treat or decrease the risk of ARDS in patients with alcoholism. We have recently shown increased levels of lung adenosine inhibit alveolar fluid clearance, an important predictor of outcome in patients with ARDS. We hypothesized that alcohol might worsen lung injury by increasing lung adenosine levels, resulting in impaired active Na(+) transport in the lung.
We treated wild-type mice with alcohol administered i.p. to achieve blood alcohol levels associated with moderate to severe intoxication and measured the rate of alveolar fluid clearance and Na,K-ATPase expression in peripheral lung tissue and assessed the effect of alcohol on survival during exposure to hyperoxia. We used primary rat alveolar type II cells to investigate the mechanisms by which alcohol regulates alveolar Na(+) transport.
Exposure to alcohol reduced alveolar fluid clearance, downregulated Na,K-ATPase in the lung tissue and worsened hyperoxia-induced lung injury. Alcohol caused an increase in BAL fluid adenosine levels. A similar increase in lung adenosine levels was observed after exposure to hyperoxia. In primary rat alveolar type II cells alcohol and adenosine decreased the abundance of the Na,K-ATPase at the basolateral membrane via a mechanism that required activation of the AMPK.
Alcohol decreases alveolar fluid clearance and impairs survival from acute lung injury. Alcohol induced increases in lung adenosine levels may be responsible for reduction in alveolar fluid clearance and associated worsening of lung injury.
PLoS ONE 01/2012; 7(1):e30448. · 4.09 Impact Factor
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István Vadász,
Laura A Dada,
Arturo Briva,
Iiro Taneli Helenius,
Kfir Sharabi,
Lynn C Welch,
Aileen M Kelly,
Benno A Grzesik, G R Scott Budinger,
Jing Liu,
Werner Seeger,
Greg J Beitel,
Yosef Gruenbaum,
Jacob I Sznajder
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ABSTRACT: Elevated CO(2) levels (hypercapnia) occur in patients with respiratory diseases and impair alveolar epithelial integrity, in part, by inhibiting Na,K-ATPase function. Here, we examined the role of c-Jun N-terminal kinase (JNK) in CO(2) signaling in mammalian alveolar epithelial cells as well as in diptera, nematodes and rodent lungs. In alveolar epithelial cells, elevated CO(2) levels rapidly induced activation of JNK leading to downregulation of Na,K-ATPase and alveolar epithelial dysfunction. Hypercapnia-induced activation of JNK required AMP-activated protein kinase (AMPK) and protein kinase C-ζ leading to subsequent phosphorylation of JNK at Ser-129. Importantly, elevated CO(2) levels also caused a rapid and prominent activation of JNK in Drosophila S2 cells and in C. elegans. Paralleling the results with mammalian epithelial cells, RNAi against Drosophila JNK fully prevented CO(2)-induced downregulation of Na,K-ATPase in Drosophila S2 cells. The importance and specificity of JNK CO(2) signaling was additionally demonstrated by the ability of mutations in the C. elegans JNK homologs, jnk-1 and kgb-2 to partially rescue the hypercapnia-induced fertility defects but not the pharyngeal pumping defects. Together, these data provide evidence that deleterious effects of hypercapnia are mediated by JNK which plays an evolutionary conserved, specific role in CO(2) signaling in mammals, diptera and nematodes.
PLoS ONE 01/2012; 7(10):e46696. · 4.09 Impact Factor
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ABSTRACT: HMG-CoA reductase inhibitors such as rosuvastatin may have immunomodulatory and anti-inflammatory effects that may reduce the severity of influenza A infection. We hypothesized that rosuvastatin would decrease viral replication, attenuate lung injury, and improve mortality following influenza A infection in mice.
C57Bl/6 mice were treated daily with rosuvastatin (10 mg/kg/day) supplemented in chow (or control chow) beginning three days prior to infection with either A//Udorn/72 [H3N2] or A/WSN/33 [H1N1] influenza A virus (1×10(5) pfu/mouse). Plaque assays were used to examine the effect of rosuvastatin on viral replication in vitro and in the lungs of infected mice. We measured cell count with differential, protein and cytokines in the bronchoalveolar lavage (BAL) fluid, histologic evidence of lung injury, and wet-to-dry ratio on Day 1, 2, 4, and 6. We also recorded daily weights and mortality.
The administration of rosuvastatin had no effect on viral clearance of influenza A after infection. Weight loss, lung inflammation and lung injury severity were similar in the rosuvastatin and control treated mice. In the mice infected with influenza A (A/WSN/33), mortality was unaffected by treatment with rosuvastatin.
Statins did not alter the replication of influenza A in vitro or enhance its clearance from the lung in vivo. Statins neither attenuated the severity of influenza A-induced lung injury nor had an effect on influenza A-related mortality. Our data suggest that the association between HMG CoA reductase inhibitors and improved outcomes in patients with sepsis and pneumonia are not attributable to their effects on influenza A infection.
PLoS ONE 01/2012; 7(4):e35788. · 4.09 Impact Factor
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Saul Soberanes,
Angel Gonzalez,
Daniela Urich,
Sergio E Chiarella,
Kathryn A Radigan,
Alvaro Osornio-Vargas,
Joy Joseph,
Balaraman Kalyanaraman,
Karen M Ridge,
Navdeep S Chandel,
Gökhan M Mutlu,
Andrea De Vizcaya-Ruiz, G R Scott Budinger
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ABSTRACT: Exposure of human populations to chronically elevated levels of ambient particulate matter air pollution < 2.5 μm in diameter (PM(2.5)) has been associated with an increase in lung cancer incidence. Over 70% of lung cancer cell lines exhibit promoter methylation of the tumor suppressor p16, an epigenetic modification that reduces its expression. We exposed mice to concentrated ambient PM(2.5) via inhalation, 8 hours daily for 3 weeks and exposed primary murine alveolar epithelial cells to daily doses of fine urban PM (5 µg/cm(2)). In both mice and alveolar epithelial cells, PM exposure increased ROS production, expression of the DNA methyltransferase 1 (DNMT1), and methylation of the p16 promoter. In alveolar epithelial cells, increased transcription of DNMT1 and methylation of the p16 promoter were inhibited by a mitochondrially targeted antioxidant and a JNK inhibitor. These findings provide a potential mechanism by which PM exposure increases the risk of lung cancer.
Scientific Reports 01/2012; 2:275.
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Matthew C Duch, G R Scott Budinger,
Yu Teng Liang,
Saul Soberanes,
Daniela Urich,
Sergio E Chiarella,
Laura A Campochiaro,
Angel Gonzalez,
Navdeep S Chandel,
Mark C Hersam,
Gökhan M Mutlu
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ABSTRACT: To facilitate the proposed use of graphene and its derivative graphene oxide (GO) in widespread applications, we explored strategies that improve the biocompatibility of graphene nanomaterials in the lung. In particular, solutions of aggregated graphene, Pluronic dispersed graphene, and GO were administered directly into the lungs of mice. The introduction of GO resulted in severe and persistent lung injury. Furthermore, in cells GO increased the rate of mitochondrial respiration and the generation of reactive oxygen species, activating inflammatory and apoptotic pathways. In contrast, this toxicity was significantly reduced in the case of pristine graphene after liquid phase exfoliation and was further minimized when the unoxidized graphene was well-dispersed with the block copolymer Pluronic. Our results demonstrate that the covalent oxidation of graphene is a major contributor to its pulmonary toxicity and suggest that dispersion of pristine graphene in Pluronic provides a pathway for the safe handling and potential biomedical application of two-dimensional carbon nanomaterials.
Nano Letters 12/2011; 11(12):5201-7. · 13.20 Impact Factor
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Gökhan M Mutlu, G R Scott Budinger,
Minghua Wu,
Anna P Lam,
Aaron Zirk,
Stephanie Rivera,
Daniela Urich,
Sergio E Chiarella,
Leonard H T Go,
Asish K Ghosh,
Moises Selman,
Annie Pardo,
John Varga,
David W Kamp,
Navdeep S Chandel,
Jacob Iasha Sznajder,
Manu Jain
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ABSTRACT: The development of organ fibrosis after injury requires activation of transforming growth factor β(1) which regulates the transcription of profibrotic genes. The systemic administration of a proteasomal inhibitor has been reported to prevent the development of fibrosis in the liver, kidney and bone marrow. It is hypothesised that proteasomal inhibition would prevent lung and skin fibrosis after injury by inhibiting TGF-β(1)-mediated transcription.
Bortezomib, a small molecule proteasome inhibitor in widespread clinical use, was administered to mice beginning 7 days after the intratracheal or intradermal administration of bleomycin and lung and skin fibrosis was measured after 21 or 40 days, respectively. To examine the mechanism of this protection, bortezomib was administered to primary normal lung fibroblasts and primary lung and skin fibroblasts obtained from patients with idiopathic pulmonary fibrosis and scleroderma, respectively.
Bortezomib promoted normal repair and prevented lung and skin fibrosis when administered beginning 7 days after the initiation of bleomycin. In primary human lung fibroblasts from normal individuals and patients with idiopathic pulmonary fibrosis and in skin fibroblasts from a patient with scleroderma, bortezomib inhibited TGF-β(1)-mediated target gene expression by inhibiting transcription induced by activated Smads. An increase in the abundance and activity of the nuclear hormone receptor PPARγ, a repressor of Smad-mediated transcription, contributed to this response.
Proteasomal inhibition prevents lung and skin fibrosis after injury in part by increasing the abundance and activity of PPARγ. Proteasomal inhibition may offer a novel therapeutic alternative in patients with dysregulated tissue repair and fibrosis.
Thorax 09/2011; 67(2):139-46. · 6.84 Impact Factor
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American Journal of Respiratory and Critical Care Medicine 09/2011; 184(5):497-8. · 11.08 Impact Factor
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Blood 09/2011; 118(9):2636-7. · 9.90 Impact Factor
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Daniela Urich,
Jessica L Eisenberg,
Kevin J Hamill,
Desire Takawira,
Sergio E Chiarella,
Saul Soberanes,
Angel Gonzalez,
Frank Koentgen,
Tomas Manghi,
Susan B Hopkinson,
Alexander V Misharin,
Harris Perlman,
Gokhan M Mutlu, G R Scott Budinger,
Jonathan C R Jones
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ABSTRACT: Laminins are heterotrimeric glycoproteins of the extracellular matrix that are secreted by epithelial cells and which are crucial for the normal structure and function of the basement membrane. We have generated a mouse harboring a conditional knockout of α3 laminin (Lama3(fl/fl)), one of the main laminin subunits in the lung basement membrane. At 60 days after intratracheal treatment of adult Lama3(fl/fl) mice with an adenovirus encoding Cre recombinase (Ad-Cre), the protein abundance of α3 laminin in whole lung homogenates was more than 50% lower than that in control-treated mice, suggesting a relatively long half-life for the protein in the lung. Upon exposure to an injurious ventilation strategy (tidal volume of 35 ml per kg of body weight for 2 hours), the mice with a knockdown of the α3 laminin subunit had less severe injury, as shown by lung mechanics, histology, alveolar capillary permeability and survival when compared with Ad-Null-treated mice. Knockdown of the α3 laminin subunit resulted in evidence of lung inflammation. However, this did not account for their resistance to mechanical ventilation. Rather, the loss of α3 laminin was associated with a significant increase in the collagen content of the lungs. We conclude that the loss of α3 laminin in the alveolar epithelium results in an increase in lung collagen, which confers resistance to mechanical injury.
Journal of Cell Science 09/2011; 124(Pt 17):2927-37. · 6.11 Impact Factor
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Qiyuan Zhou,
Annie Pardo,
Melanie Königshoff,
Oliver Eickelberg, G R Scott Budinger,
Krishna Thavarajah,
Cara J Gottardi,
Jonathan Jones,
John Varga,
Moises Selman,
Jacob I Sznajder,
J Usha Raj,
Guofei Zhou
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ABSTRACT: Idiopathic pulmonary fibrosis (IPF) is characterized by exaggerated fibroblast proliferation and accumulation of collagens and fibronectin. The extracellular fibronectin and collagen network is regulated by von Hippel-Lindau protein (pVHL). However, it is unknown whether pVHL contributes to pulmonary fibrosis. We found that lungs from patients with IPF expressed increased levels of pVHL in fibroblastic foci. Bleomycin treatment also induced pVHL in lung fibroblasts, but not in alveolar type II cells. Overexpression of pVHL increased lung fibroblast proliferation, protein abundance of fibronectin and collagen, and extracellular fibronectin. In addition, overexpression of pVHL induced expression of the α5 integrin subunit. Overexpression of pVHL did not alter hypoxia-inducible factor luciferase reporter activity and mRNA expression of vascular endothelial growth factor. Fibroblasts overexpressing pVHL were more sensitive to RGD peptide-mediated reduction in proliferation. Activating α5 and β1 integrin increased proliferation of fibroblasts overexpressing pVHL and those cells were more resistant to the inhibition of α5 integrin. Overexpression of pVHL also increased activation of focal adhesion kinase (FAK). Moreover, suppression of pVHL prevented TGF-β1-induced proliferation of mouse embryonic fibroblasts. Taken together, our results indicate that elevated expression of pVHL results in the aberrant fibronectin expression, activation of integrin/FAK signaling, fibroblast proliferation, and fibrosis.
The FASEB Journal 06/2011; 25(9):3032-44. · 5.71 Impact Factor
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American Journal of Respiratory and Critical Care Medicine 06/2011; 183(12):1614-9. · 11.08 Impact Factor
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ABSTRACT: Pulmonary fibrosis is a disease that results in loss of normal lung architecture, but the signaling events that drive tissue destruction are incompletely understood. Wnt/β-catenin signaling is important in normal lung development, but whether abnormal signaling occurs in lung fibrosis due to systemic sclerosis and the consequences of β-catenin signaling toward the fibrogenic phenotype remain poorly defined. In this study, we show nuclear β-catenin accumulation in fibroblastic foci from lungs of patients with systemic sclerosis-associated advanced pulmonary fibrosis. Forced activation of β-catenin signaling in three independently derived sources of normal human lung fibroblasts promotes proliferation and migratory activities but is not sufficient to activate classic markers of fibroblast activation, such as TGF-β, type 1 collagen, α-smooth muscle actin, and connective tissue growth factor. These findings indicate that activation of β-catenin signaling in pulmonary fibroblasts may be a common feature of lung fibrosis, contributing to fibroproliferative and migratory activities associated with the disease.
American Journal of Respiratory Cell and Molecular Biology 03/2011; 45(5):915-22. · 5.13 Impact Factor
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ABSTRACT: In alveolar epithelial cells (AECs), the membrane-anchored proteoglycan dystroglycan (DG) is a mechanoreceptor that transmits mechanical stretch forces to activate independently the ERK1/2 and the adenosine 5'-monophosphate-activated protein kinase (AMPK) signaling cascades in a process called pathway bifurcation. We tested the hypothesis that the cytoskeleton cross-linker plectin, known to bind both DG and AMPK in muscle cells, acts as a scaffold to regulate DG-mediated mechanical stimulation and pathway bifurcation. We demonstrate that plectin and DG form a complex in AECs and that this complex interacts with ERK1/2 and AMPK. Plectin knockdown reduces DG interaction with AMPK but not with ERK1/2. Despite this, mechanoactivation of both signaling pathways is significantly attenuated in AECs deficient in plectin. Thus, DG has the dual role of mechanical receptor and scaffold for ERK1/2, whereas plectin acts as a scaffold for AMPK signaling but is also required for DG-mediated ERK1/2 activation. We conclude that the DG-plectin complex plays a central role in transmitting mechanical stress from the extracellular matrix to the cytoplasm.
Journal of Biological Chemistry 02/2011; 286(8):6301-10. · 4.77 Impact Factor
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G R Scott Budinger,
Joanne L McKell,
Daniela Urich,
Nancy Foiles,
Ivy Weiss,
Sergio E Chiarella,
Angel Gonzalez,
Saul Soberanes,
Andrew J Ghio,
Recep Nigdelioglu,
Ece A Mutlu,
Kathryn A Radigan,
David Green,
Hau C Kwaan,
Gökhan M Mutlu
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ABSTRACT: Exposure of human populations to ambient particulate matter (PM) air pollution significantly contributes to the mortality attributable to ischemic cardiovascular events. We reported that mice treated with intratracheally instilled PM develop a prothrombotic state that requires the release of IL-6 by alveolar macrophages. We sought to determine whether exposure of mice to PM increases the levels of PAI-1, a major regulator of thrombolysis, via a similar or distinct mechanism.
Adult, male C57BL/6 and IL-6 knock out (IL-6(-/-)) mice were exposed to either concentrated ambient PM less than 2.5 µm (CAPs) or filtered air 8 hours daily for 3 days or were exposed to either urban particulate matter or PBS via intratracheal instillation and examined 24 hours later. Exposure to CAPs or urban PM resulted in the IL-6 dependent activation of coagulation in the lung and systemically. PAI-1 mRNA and protein levels were higher in the lung and adipose tissue of mice treated with CAPs or PM compared with filtered air or PBS controls. The increase in PAI-1 was similar in wild-type and IL-6(-/-) mice but was absent in mice treated with etanercept, a TNF-α inhibitor. Treatment with etanercept did not prevent the PM-induced tendency toward thrombus formation.
Mice exposed to inhaled PM exhibited a TNF-α-dependent increase in PAI-1 and an IL-6-dependent activation of coagulation. These results suggest that multiple mechanisms link PM-induced lung inflammation with the development of a prothrombotic state.
PLoS ONE 01/2011; 6(4):e18525. · 4.09 Impact Factor
