Daniela Urich

Minimizing Oxidation and Stable Nanoscale Dispersion Improves the Biocompatibility of Graphene in the Lung

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.

Biocompatible nanoscale dispersion of single-walled carbon nanotubes minimizes in vivo pulmonary toxicity.

Excitement surrounding the attractive physical and chemical characteristics of single walled carbon nanotubes (SWCNTs) has been tempered by concerns regarding their potential health risks. Here we consider the lung toxicity of nanoscale dispersed SWCNTs (mean diameter approximately 1 nm). Because dispersion of the SWCNTs increases their aspect ratio relative to as-produced aggregates, we directly test the prevailing hypothesis that lung toxicity associated with SWCNTs compared with other carbon structures is attributable to the large aspect ratio of the individual particles. Thirty days after their intratracheal administration to mice, the granuloma-like structures with mild fibrosis in the large airways observed in mice treated with aggregated SWCNTs were absent in mice treated with nanoscale dispersed SWCNTs. Examination of lung sections from mice treated with nanoscale dispersed SWCNTs revealed uptake of the SWCNTs by macrophages and gradual clearance over time. We conclude that the toxicity of SWCNTs in vivo is attributable to aggregation of the nanomaterial rather than the large aspect ratio of the individual nanotubes. Biocompatible nanoscale dispersion provides a scalable method to generate purified preparations of SWCNTs with minimal toxicity, thus allowing them to be used safely in commercial and biomedical applications.

Mitochondrial Complex III-generated Oxidants Activate ASK1 and JNK to Induce Alveolar Epithelial Cell Death following Exposure to Particulate Matter Air Pollution

We have previously reported that airborne particulate matter air pollution (PM) activates the intrinsic apoptotic pathway in alveolar epithelial cells through a pathway that requires the mitochondrial generation of reactive oxygen species (ROS) and the activation of p53. We sought to examine the source of mitochondrial oxidant production and the molecular links between ROS generation and the activation of p53 in response to PM exposure. Using a mitochondrially targeted ratiometric sensor (Ro-GFP) in cells lacking mitochondrial DNA (ρ0 cells) and cells stably expressing a small hairpin RNA directed against the Rieske iron-sulfur protein, we show that site III of the mitochondrial electron transport chain is primarily responsible for fine PM (PM2.5)-induced oxidant production. In alveolar epithelial cells, the overexpression of SOD1 prevented the PM2.5-induced ROS generation from the mitochondria and prevented cell death. Infection of mice with an adenovirus encoding SOD1 prevented the PM2.5-induced death of alveolar epithelial cells and the associated increase in alveolar-capillary permeability. Treatment with PM2.5 resulted in the ROS-mediated activation of the oxidant-sensitive kinase ASK1 and its downstream kinase JNK. Murine embryonic fibroblasts from ASK1 knock-out mice, alveolar epithelial cells transfected with dominant negative constructs against ASK1, and pharmacologic inhibition of JNK with SP600125 (25 μm) prevented the PM2.5-induced phosphorylation of p53 and cell death. We conclude that particulate matter air pollution induces the generation of ROS primarily from site III of the mitochondrial electron transport chain and that these ROS activate the intrinsic apoptotic pathway through ASK1, JNK, and p53.

Particulate Matter-Induced Lung Inflammation Increases Systemic Levels of PAI-1 and Activates Coagulation Through Distinct Mechanisms

Background 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. Methods and Principal Findings 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. Conclusions 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.

PAI-1–regulated extracellular proteolysis governs senescence and survival in Klotho mice

Significance Plasminogen activator inhibitor-1 (PAI-1) is an essential mediator of cellular senescence in vitro and is one of the biochemical fingerprints of senescence in vivo. Klotho-deficient (kl/kl) mice display a complex phenotype reminiscent of human aging and exhibit age-dependent increases in PAI-1 in tissues and in plasma. Thus, we hypothesized that PAI-1 contributes to the aging-like phenotype of kl/kl mice. We observed that either genetic deficiency or pharmacological inhibition of PAI-1 in kl/kl mice was associated with reduced evidence of senescence, preserved organ structure and function, and a fourfold increase in median lifespan. These findings indicate that PAI-1 is a critical mediator of senescence in vivo and defines a novel target for the prevention and treatment of age-related disorders in man. Cellular senescence restricts the proliferative capacity of cells and is accompanied by the production of several proteins, collectively termed the “senescence-messaging secretome” (SMS). As senescent cells accumulate in tissue, local effects of the SMS have been hypothesized to disrupt tissue regenerative capacity. Klotho functions as an aging-suppressor gene, and Klotho-deficient (kl/kl) mice exhibit an accelerated aging-like phenotype that includes a truncated lifespan, arteriosclerosis, and emphysema. Because plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor (SERPIN), is elevated in kl/kl mice and is a critical determinant of replicative senescence in vitro, we hypothesized that a reduction in extracellular proteolytic activity contributes to the accelerated aging-like phenotype of kl/kl mice. Here we show that PAI-1 deficiency retards the development of senescence and protects organ structure and function while prolonging the lifespan of kl/kl mice. These findings indicate that a SERPIN-regulated cell-nonautonomous proteolytic cascade is a critical determinant of senescence in vivo.

Vimentin regulates activation of the NLRP3 inflammasome.

Activation of the NLRP3 inflammasome and subsequent maturation of IL-1β have been implicated in acute lung injury (ALI), resulting in inflammation and fibrosis. We investigated the role of vimentin, a type III intermediate filament, in this process using three well-characterized murine models of ALI known to require NLRP3 inflammasome activation. We demonstrate that central pathophysiologic events in ALI (inflammation, IL-1β levels, endothelial and alveolar epithelial barrier permeability, remodelling and fibrosis) are attenuated in the lungs of Vim−/− mice challenged with LPS, bleomycin and asbestos. Bone marrow chimeric mice lacking vimentin have reduced IL-1β levels and attenuated lung injury and fibrosis following bleomycin exposure. Furthermore, decreased active caspase-1 and IL-1β levels are observed in vitro in Vim−/− and vimentin-knockdown macrophages. Importantly, we show direct protein–protein interaction between NLRP3 and vimentin. This study provides insights into lung inflammation and fibrosis and suggests that vimentin may be a key regulator of the NLRP3 inflammasome. Supplementary information The online version of this article (doi:10.1038/ncomms7574) contains supplementary material, which is available to authorized users.

Particulate matter Air Pollution induces hypermethylation of the p16 promoter Via a mitochondrial ROS-JNK-DNMT1 pathway

Exposure of human populations to chronically elevated levels of ambient particulate matter air pollution < 2.5 μm in diameter (PM2.5) 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 PM2.5 via inhalation, 8u2005hours daily for 3 weeks and exposed primary murine alveolar epithelial cells to daily doses of fine urban PM (5 µg/cm2). 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.

Epithelial Cell Death Is an Important Contributor to Oxidant-mediated Acute Lung Injury

RATIONALEnAcute lung injury and the acute respiratory distress syndrome are characterized by increased lung oxidant stress and apoptotic cell death. The contribution of epithelial cell apoptosis to the development of lung injury is unknown.nnnOBJECTIVESnTo determine whether oxidant-mediated activation of the intrinsic or extrinsic apoptotic pathway contributes to the development of acute lung injury.nnnMETHODSnExposure of tissue-specific or global knockout mice or cells lacking critical components of the apoptotic pathway to hyperoxia, a well-established mouse model of oxidant-induced lung injury, for measurement of cell death, lung injury, and survival.nnnMEASUREMENTS AND MAIN RESULTSnWe found that the overexpression of SOD2 prevents hyperoxia-induced BAX activation and cell death in primary alveolar epithelial cells and prolongs the survival of mice exposed to hyperoxia. The conditional loss of BAX and BAK in the lung epithelium prevented hyperoxia-induced cell death in alveolar epithelial cells, ameliorated hyperoxia-induced lung injury, and prolonged survival in mice. By contrast, Cyclophilin D-deficient mice were not protected from hyperoxia, indicating that opening of the mitochondrial permeability transition pore is dispensable for hyperoxia-induced lung injury. Mice globally deficient in the BH3-only proteins BIM, BID, PUMA, or NOXA, which are proximal upstream regulators of BAX and BAK, were not protected against hyperoxia-induced lung injury suggesting redundancy of these proteins in the activation of BAX or BAK.nnnCONCLUSIONSnMitochondrial oxidant generation initiates BAX- or BAK-dependent alveolar epithelial cell death, which contributes to hyperoxia-induced lung injury.

Leptin Promotes Fibroproliferative Acute Respiratory Distress Syndrome by Inhibiting Peroxisome Proliferator–activated Receptor-γ

RATIONALEnDiabetic patients have a lower incidence of acute respiratory distress syndrome (ARDS), and those who develop ARDS are less likely to die. The mechanisms that underlie this protection are unknown.nnnOBJECTIVESnTo determine whether leptin resistance, a feature of diabetes, prevents fibroproliferation after lung injury.nnnMETHODSnWe examined lung injury and fibroproliferation after the intratracheal instillation of bleomycin in wild-type and leptin-resistant (db/db) diabetic mice. We examined the effect of leptin on transforming growth factor (TGF)-β(1)-mediated transcription in primary normal human lung fibroblasts. Bronchoalveolar lavage fluid (BAL) samples from patients with ARDS and ventilated control subjects were obtained for measurement of leptin and active TGF-β(1) levels.nnnMEASUREMENTS AND MAIN RESULTSnDiabetic mice (db/db) were resistant to lung fibrosis. The db/db mice had higher levels of peroxisome proliferator-activated receptor-γ (PPARγ), an inhibitor of the transcriptional response to TGF-β(1), a cytokine critical in the pathogenesis of fibroproliferative ARDS. In normal human lung fibroblasts, leptin augmented the transcription of profibrotic genes in response to TGF-β(1) through a mechanism that required PPARγ. In patients with ARDS, BAL leptin levels were elevated and correlated with TGF-β(1) levels. Overall, there was no significant relationship between BAL leptin levels and clinical outcomes; however, in nonobese patients, higher BAL leptin levels were associated with fewer intensive care unit- and ventilator-free days and higher mortality.nnnCONCLUSIONSnLeptin signaling is required for bleomycin-induced lung fibrosis. Leptin augments TGF-β(1) signaling in lung fibroblasts by inhibiting PPARγ. These findings provide a mechanism for the observed protection against ARDS observed in diabetic patients.

Proteasomal inhibition after injury prevents fibrosis by modulating TGF-β 1 signalling

Background The development of organ fibrosis after injury requires activation of transforming growth factor β1 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-β1-mediated transcription. Methods Bortezomib, a small molecule proteasome inhibitor in widespread clinical use, was administered to mice beginning 7u2005days after the intratracheal or intradermal administration of bleomycin and lung and skin fibrosis was measured after 21 or 40u2005days, 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. Results Bortezomib promoted normal repair and prevented lung and skin fibrosis when administered beginning 7u2005days 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-β1-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. Conclusions 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.