Background Epithelial cell death is a major contributor to fibrogenesis in the lung. In this study, we sought to determine the function of mitochondria and their clearance (mitophagy) in alveolar epithelial cell death and fibrosis. Methods We studied markers of mitochondrial injury and the mitophagy marker, PTEN-induced putative kinase 1 (PINK1), in IPF lung tissues by Western blotting, transmission electron microscopy (TEM), and immunofluorescence. In vitro experiments were carried out in lung epithelial cells stimulated with transforming growth factor-β1 (TGF-β1). Changes in cell function were measured by Western blotting, flow cytometry and immunofluorescence. In vivo experiments were performed using the murine bleomycin model of lung fibrosis. Results Evaluation of IPF lung tissue demonstrated increased PINK1 expression by Western blotting and immunofluorescence and increased numbers of damaged mitochondria by TEM. In lung epithelial cells, TGF-β1 induced mitochondrial depolarization, mitochondrial ROS, and PINK1 expression; all were abrogated by mitochondrial ROS scavenging. Finally, Pink1-/- mice were more susceptible than control mice to bleomycin induced lung fibrosis. Conclusion TGF-β1 induces lung epithelial cell mitochondrial ROS and depolarization and stabilizes the key mitophagy initiating protein, PINK1. PINK1 ameliorates epithelial cell death and may be necessary to limit fibrogenesis.

Despite public health campaigns discouraging smoking, 1,000 American children every day become smokers, ensuring that tobacco-related health complications will be with us for decades to come. Smoking is the greatest risk factor for both chronic obstructive lung disease and interstitial lung disease. The facts that not every smoker develops chronic lung disease and that lung pathology differs markedly among smokers indicate that individual susceptibility must be a central determinant of lung injury responses to cigarette smoke. Comparative examination of pathogenic mechanisms of smoke-induced lung disease can shed light on the homeostatic pathways critical to maintaining lung health. In this review, we explore common and divergent biological forces tilting the lung homeostatic balance away from health and toward emphysema or pulmonary fibrosis. We emphasize recent insights that highlight the greatest contrasts or similarities in the pathogenesis of these two chronic lung disease phenotypes.

Vascular endothelial growth factor (VEGF)-D, a member of the VEGF family, induces both angiogenesis and lymphangiogenesis by activating VEGF receptor-2 (VEGFR-2) and VEGFR-3 on the surface of endothelial cells. Transforming growth factor (TGF)-β1 has been shown to stimulate VEGF-A expression in human lung fibroblast via the Smad3 signaling pathway and to induce VEGF-C in human proximal tubular epithelial cells. However, the effects of TGF-β1 on VEGF-D regulation are unknown. To investigate the regulation of VEGF-D, human lung fibroblasts were studied under pro-fibrotic conditions in vitro and in idiopathic pulmonary fibrosis (IPF) lung tissue. We demonstrate that TGF-β1 downregulates VEGF-D expression in a dose- and time-dependent manner in human lung fibroblasts. This TGF-β1 effect can be abolished by inhibitors of TGF-β type I receptor kinase and Jun NH2-terminal kinase (JNK), but not by Smad3 knockdown. In addition, VEGF-D knockdown in human lung fibroblasts induces G1/S transition and promotes cell proliferation. Importantly, VEGF-D protein expression is decreased in lung homogenates from IPF patients compared with control lung. In IPF lung sections, fibroblastic foci show very weak VEGF-D immunoreactivity, whereas VEGF-D is abundantly expressed within alveolar interstitial cells in control lung. Taken together, our data identify a novel mechanism for downstream signal transduction induced by TGF-β1 in lung fibroblasts, through which they may mediate tissue remodeling in IPF.

RATIONALE:Alveolar transforming growth factor (TGF)-β1 signaling and expression of TGF-β1 target genes are increased in patients with idiopathic pulmonary fibrosis (IPF) and in animal models of pulmonary fibrosis. Internalization and degradation of TGF-β receptor TβRI inhibits TGF-β signaling and could attenuate development of experimental lung fibrosis.OBJECTIVES:To demonstrate that after experimental lung injury, human syndecan-2 confers antifibrotic effects by inhibiting TGF-β1 signaling in alveolar epithelial cells.METHODS:Microarray assays were performed to identify genes differentially expressed in alveolar macrophages of patients with IPF versus control subjects. Transgenic mice that constitutively overexpress human syndecan-2 in macrophages were developed to test the antifibrotic properties of syndecan-2. In vitro assays were performed to determine syndecan-2-dependent changes in epithelial cell TGF-β1 signaling, TGF-β1, and TβRI internalization and apoptosis. Wild-type mice were treated with recombinant human syndecan-2 during the fibrotic phase of bleomycin-induced lung injury.MEASUREMENTS AND MAIN RESULTS:We observed significant increases in alveolar macrophage syndecan-2 levels in patients with IPF. Macrophage-specific overexpression of human syndecan-2 in transgenic mice conferred antifibrotic effects after lung injury by inhibiting TGF-β1 signaling and downstream expression of TGF-β1 target genes, reducing extracellular matrix production and alveolar epithelial cell apoptosis. In vitro, syndecan-2 promoted caveolin-1-dependent internalization of TGF-β1 and TβRI in alveolar epithelial cells, which inhibited TGF-β1 signaling and epithelial cell apoptosis. Therapeutic administration of human syndecan-2 abrogated lung fibrosis in mice.CONCLUSIONS:Alveolar macrophage syndecan-2 exerts antifibrotic effects by promoting caveolin-1-dependent TGF-β1 and TβRI internalization and inhibiting TGF-β1 signaling in alveolar epithelial cells. Hence, molecules that facilitate TβRI degradation via endocytosis represent potential therapies for pulmonary fibrosis.

Rationale: Retinoid-related orphan receptor-alpha (RORa) is expressed at high levels in the lungs of patients with COPD. We investigate the role of RORa in promoting autophagy, a recently described pathogenic mechanism for the promotion of emphysema. Methods: Western blot was used to assess protein expression in cells and lung tissue. Co-immunoprecipitation and confocal microscopy were used to evaluate RORa-p53 interactions. A RORa expression plasmid was used to induce autophagy in vitro. Results: In response to cigarette smoke extract (CSE), RORa deficient cells exhibit fewer autophagosomes, lower levels of LC3II, lower levels of autophagic flux and higher levels of p62 than control cells. Conversely, overexpression of RORa in vitro induces markers of autophagy independent of other stimuli. RORa expression is not induced by rapamycin treatment, and pS6 levels are unaffected by the absence of RORa. RORa co-immunoprecipitates with p53 and localizes to the nucleus with p53. A p53 reporter shows marked downregulation of p53 transcription activity in RORa null cells following treatment with CSE. DRAM, a p53-induced modulator of autophagy, is expressed at significantly lower levels in RORa null cells compared with wild type cells. Conclusion: Our findings suggest that RORa promotes autophagy and that autophagy is impaired in the absence of RORa. This occurs via non-mTORC1 dependent mechanisms, most likely by induction of the p53-induced modulator of autophagy, DRAM. Rationale: Retinoid-related orphan receptor-alpha (RORa) is expressed at high levels in the lungs of patients with COPD. We investigate the role of RORa in promoting autophagy, a recently described pathogenic mechanism for the promotion of emphysema. Methods: Western blot was used to assess protein expression in cells and lung tissue. Co-immunoprecipitation and confocal microscopy were used to evaluate RORa-p53 interactions. A RORa expression plasmid was used to induce autophagy in vitro. Results: In response to cigarette smoke extract (CSE), RORa deficient cells exhibit fewer autophagosomes, lower levels of LC3II, lower levels of autophagic flux and higher levels of p62 than control cells. Conversely, overexpression of RORa in vitro induces markers of autophagy independent of other stimuli. RORa expression is not induced by rapamycin treatment, and pS6 levels are unaffected by the absence of RORa. RORa co-immunoprecipitates with p53 and localizes to the nucleus with p53. A p53 reporter shows marked downregulation of p53 transcription activity in RORa null cells following treatment with CSE. DRAM, a p53-induced modulator of autophagy, is expressed at significantly lower levels in RORa null cells compared with wild type cells. Conclusion: Our findings suggest that RORa promotes autophagy and that autophagy is impaired in the absence of RORa. This occurs via non-mTORC1 dependent mechanisms, most likely by induction of the p53-induced modulator of autophagy, DRAM.

Autophagy is a homeostatic process common to all eukaryotic cells that serves to degrade intracellular components. Among three classes of autophagy, macroautophagy is best understood, and is the subject of this Review. The function of autophagy is multifaceted, and includes removal of long-lived proteins and damaged or unneeded organelles, recycling of intracellular components for nutrients, and defense against pathogens. This process has been extensively studied in yeast, and understanding of its functional significance in human disease is also increasing. This Review explores the basic machinery and regulation of autophagy in mammalian systems, methods employed to measure autophagic activity, and then focuses on recent discoveries about the functional significance of autophagy in respiratory diseases, including chronic obstructive pulmonary disease, cystic fibrosis, tuberculosis, idiopathic pulmonary fibrosis, pulmonary arterial hypertension, acute lung injury, and lymphangioleiomyomatosis.

Luciferases have proven to be useful tools in advancing our understanding of biologic processes. Having a multitude of bioluminescent reporters with different properties is highly desirable. We characterized codon-optimized thermostable green- and red-emitting luciferase variants from the Italian firefly Luciola italica for mammalian gene expression in culture and in vivo. Using lentivirus vectors to deliver and stably express these luciferases in mammalian cells, we showed that both variants displayed similar levels of activity and protein half-lives as well as similar light emission kinetics and higher stability compared to the North American firefly luciferase. Further, we characterized the red-shifted variant for in vivo bioluminescence imaging. Intramuscular injection of tumor cells stably expressing this variant into nude mice yielded a robust luciferase activity. Light emission peaked at 10 minutes post-d-luciferin injection and retained > 60% of signal at 1 hour. Similarly, luciferase activity from intracranially injected glioma cells expressing the red-shifted variant was readily detected and used as a marker to monitor tumor growth over time. Overall, our characterization of these codon-optimized luciferases lays the groundwork for their further use as bioluminescent reporters in mammalian cells.

Background Autophagy is a basic cellular homeostatic process important to cell fate decisions under conditions of stress. Dysregulation of autophagy impacts numerous human diseases including cancer and chronic obstructive lung disease. This study investigates the role of autophagy in idiopathic pulmonary fibrosis. Methods Human lung tissues from patients with IPF were analyzed for autophagy markers and modulating proteins using western blotting, confocal microscopy and transmission electron microscopy. To study the effects of TGF-β1 on autophagy, human lung fibroblasts were monitored by fluorescence microscopy and western blotting. In vivo experiments were done using the bleomycin-induced fibrosis mouse model. Results Lung tissues from IPF patients demonstrate evidence of decreased autophagic activity as assessed by LC3, p62 protein expression and immunofluorescence, and numbers of autophagosomes. TGF-β1 inhibits autophagy in fibroblasts in vitro at least in part via activation of mTORC1; expression of TIGAR is also increased in response to TGF-β1. In the bleomycin model of pulmonary fibrosis, rapamycin treatment is antifibrotic, and rapamycin also decreases expression of á-smooth muscle actin and fibronectin by fibroblasts in vitro. Inhibition of key regulators of autophagy, LC3 and beclin-1, leads to the opposite effect on fibroblast expression of á-smooth muscle actin and fibronectin. Conclusion Autophagy is not induced in pulmonary fibrosis despite activation of pathways known to promote autophagy. Impairment of autophagy by TGF-β1 may represent a mechanism for the promotion of fibrogenesis in IPF.

RATIONALE:The discovery that retinoic acid-related orphan receptor (Rora)-α is highly expressed in lungs of patients with COPD led us to hypothesize that Rora may contribute to the pathogenesis of emphysema.OBJECTIVES:To determine the role of Rora in smoke-induced emphysema.METHODS:Cigarette smoke extract in vitro and elastase or cigarette smoke exposure in vivo were used to model smoke-related cell stress and airspace enlargement. Lung tissue from patients undergoing lung transplantation was examined for markers of DNA damage and Rora expression.MEASUREMENTS AND MAIN RESULTS:Rora expression was induced by cigarette smoke in mice and in cell culture. Gene expression profiling of Rora-null mice exposed to cigarette smoke demonstrated enrichment for genes involved in DNA repair. Rora expression increased and Rora translocated to the nucleus after DNA damage. Inhibition of ataxia telangiectasia mutated decreased the induction of Rora. Gene silencing of Rora attenuated apoptotic cell death in response to cigarette smoke extract, whereas overexpression of Rora enhanced apoptosis. Rora-deficient mice were protected from elastase and cigarette smoke induced airspace enlargement. Finally, lungs of patients with COPD showed evidence of increased DNA damage even in the absence of active smoking.CONCLUSIONS:Taken together, these findings suggest that DNA damage may contribute to the pathogenesis of emphysema, and that Rora has a previously unrecognized role in cellular responses to genotoxicity. These findings provide a potential link between emphysema and features of premature ageing, including enhanced susceptibility to lung cancer.

To the editor: Luciferases have played an integral role in bioluminescence imaging as useful tools in monitoring various biological processes.1Badr CE Tannous BA Bioluminescence imaging: progress and applications.Trends Biotechnol. 2011; 29: 624-633Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar,2Prescher JA Contag CH Guided by the light: visualizing biomolecular processes in living animals with bioluminescence.Curr Opin Chem Biol. 2010; 14: 80-89Crossref PubMed Scopus (203) Google Scholar,3Weissleder R Pittet MJ Imaging in the era of molecular oncology.Nature. 2008; 452: 580-589Crossref PubMed Scopus (1980) Google Scholar The most commonly used luciferase is from the American firefly Photinus pyralis (Fluc), which uses beetle D-luciferin as a substrate.4Pichler A Prior JL Luker GD Piwnica-Worms D Generation of a highly inducible Gal4®Fluc universal reporter mouse for in vivo bioluminescence imaging.Proc Natl Acad Sci USA. 2008; 105: 15932-15937Crossref PubMed Scopus (32) Google Scholar,5Rabinovich BA Ye Y Etto T Chen JQ Levitsky HI Overwijk WW et al.Visualizing fewer than 10 mouse T cells with an enhanced firefly luciferase in immunocompetent mouse models of cancer.Proc Natl Acad Sci USA. 2008; 105: 14342-14346Crossref PubMed Scopus (166) Google Scholar Other promising luciferases for in vivo imaging are from coelenterates such as the sea pansy Renilla reniformis (Rluc) and the marine copepod Gaussia princeps (Gluc).6Loening AM Dragulescu-Andrasi A Gambhir SS A red-shifted Renilla luciferase for transient reporter-gene expression.Nat Methods. 2010; 7: 5-6Crossref PubMed Scopus (72) Google Scholar,7Santos EB Yeh R Lee J Nikhamin Y Punzalan B Punzalan B et al.Sensitive in vivo imaging of T cells using a membrane-bound Gaussia princeps luciferase.Nat Med. 2009; 15: 338-344Crossref PubMed Scopus (105) Google Scholar,8Stefan E Aquin S Berger N Landry CR Nyfeler B Bouvier M et al.Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo.Proc Natl Acad Sci USA. 2007; 104: 16916-16921Crossref PubMed Scopus (155) Google Scholar,9Tannous BA Kim DE Fernandez JL Weissleder R Breakefield XO Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo.Mol Ther. 2005; 11: 435-443Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar,10Wurdinger T Badr C Pike L de Kleine R Weissleder R Breakefield XO et al.A secreted luciferase for ex vivo monitoring of in vivo processes.Nat Methods. 2008; 5: 171-173Crossref PubMed Scopus (232) Google Scholar Both of these luciferases catalyze the oxidative decarboxylation of their substrate coelenterazine (CTZ), resulting in emission of blue light.1Badr CE Tannous BA Bioluminescence imaging: progress and applications.Trends Biotechnol. 2011; 29: 624-633Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar Because Fluc and Gluc/Rluc utilize different substrates, they can be combined as dual reporters for monitoring two distinct biological processes.11Bhaumik S Gambhir SS Optical imaging of Renilla luciferase reporter gene expression in living mice.Proc Natl Acad Sci USA. 2002; 99: 377-382Crossref PubMed Scopus (474) Google Scholar,12Wang H Cao F De A Contag C Gambhir DD Wu JC et al.Trafficking mesenchymal stem cell engraftment and differentiation in tumor-bearing mice by bioluminescence imaging.Stem Cells. 2009; 27: 1548-1558Crossref PubMed Scopus (189) Google Scholar Firefly, Gaussia, and Renilla luciferases are being used as reporters for the in vivo monitoring of numerous biological processes in a variety of fields, including immunology, oncology, virology, neuroscience, and gene and cell therapy.1Badr CE Tannous BA Bioluminescence imaging: progress and applications.Trends Biotechnol. 2011; 29: 624-633Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar,2Prescher JA Contag CH Guided by the light: visualizing biomolecular processes in living animals with bioluminescence.Curr Opin Chem Biol. 2010; 14: 80-89Crossref PubMed Scopus (203) Google Scholar,3Weissleder R Pittet MJ Imaging in the era of molecular oncology.Nature. 2008; 452: 580-589Crossref PubMed Scopus (1980) Google Scholar The major drawback to using coelenterate luciferases as reporters for in vivo imaging has been the unavailability of a CTZ variant that is soluble in aqueous solution, making the use of alcohol essential in animals. The ensuing toxicity/solubility in vivo limits the injected dose (typically up to 100 μg), leading to lower sensitivity. Here, we show that a water-soluble coelenterazine (s-CTZ) that was recently synthesized and is commercially available yields up to 100-fold greater sensitivity as compared with native CTZ for in vivo imaging of Renilla and Gaussia luciferases. This s-CTZ is very useful for monitoring in vivo biological processes in which high sensitivity is required, such as detecting few circulating cells, early tumor metastasis, and apoptosis.7Santos EB Yeh R Lee J Nikhamin Y Punzalan B Punzalan B et al.Sensitive in vivo imaging of T cells using a membrane-bound Gaussia princeps luciferase.Nat Med. 2009; 15: 338-344Crossref PubMed Scopus (105) Google Scholar,10Wurdinger T Badr C Pike L de Kleine R Weissleder R Breakefield XO et al.A secreted luciferase for ex vivo monitoring of in vivo processes.Nat Methods. 2008; 5: 171-173Crossref PubMed Scopus (232) Google Scholar,13Chung E Yamashita H Au P Tannous BA Fukumura D Jain RK Secreted Gaussia luciferase as a biomarker for monitoring tumor progression and treatment response of systemic metastases.PLoS One. 2009; 4: e8316Crossref PubMed Scopus (71) Google Scholar,14Niers JM Kerami M Pike L Lewandrowski G Tannous BA Multimodal in vivo imaging and blood monitoring of intrinsic and extrinsic apoptosis.Mol Ther. 2011; 19: 1090-1096Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar We first compared the bioluminescence reaction kinetics for coelenterate luciferases using either CTZ or s-CTZ. We determined the light output for Gluc and Rluc samples over time using a spectrophotometer (Supplementary Methods online). Both substrates showed similar flash-type bioluminescence reactions for Gluc and Rluc (Supplementary Figure S1a). Recently, we characterized a Gluc variant (GlucM43I) that catalyzes a glow-type bioluminescence reaction in the presence of a detergent such as Triton X-100 (15Maguire CA Deliolanis NC Pike L Niers JM Tjon-Kon-Fat LA Sena-Esteves M et al.Gaussia luciferase variant for high-throughput functional screening applications.Anal Chem. 2009; 81: 7102-7106Crossref PubMed Scopus (65) Google Scholar). We compared the reaction kinetics of this variant as well as wild-type Gluc in the presence of Triton X-100 using CTZ and s-CTZ. The substrates exhibited similar glow-type reactions with GlucM43I and enhanced stable light output with wild-type Gluc in the presence of Triton X-100 (Supplementary Figure S1a). We also investigated whether s-CTZ has any influence on the characteristics of light emission by Gluc or Rluc. We analyzed their emission spectra using either CTZ or s-CTZ and observed that both luciferases exhibited their characteristic peaks around 480 nm, indicating that the water-soluble substrate had no effect on the light emission of coelenterate luciferases (Supplementary Figure S1b). These data suggest that the s-CTZ yields bioluminescence characteristics similar to those of native CTZ. We then determined the effect of native and water-soluble CTZ concentrations on Gluc and Rluc light production. Gli36 human glioma cells were transduced with a lentivirus vector to express either Rluc or Gluc (Supplementary Methods). Equal aliquots of conditioned medium (for Gluc) or cell lysates (for Rluc) were analyzed with increasing doses of both CTZs using a luminometer. As expected, increasing the substrate dose increased the signal from Gluc in a roughly linear manner, whereas Rluc reached a plateau at around 2 μg/ml. Interestingly, s-CTZ yielded over 10-fold higher activity for Gluc and fourfold for Rluc at all concentrations tested, as compared with CTZ (Supplementary Figure S2a). We also compared the sensitivity of the water-soluble CTZ to the native substrate in detecting viability of mammalian cells. Different numbers of Gli36 glioma cells stably expressing Gluc or Rluc were plated in 96-well plates. Twenty-four hours later, an aliquot of the conditioned medium (for Gluc) or total cell lysate (for Rluc) was assayed using both CTZs. Again, we observed over 10-fold higher photon output with Gluc and fourfold for Rluc using s-CTZ, as compared with the native CTZ substrate. These data collectively suggest that the water-soluble CTZ could be more sensitive for detecting coelenterate luciferases in vitro and in vivo (Supplementary Figure S2b). Finally, we determined the usefulness of s-CTZ for in vivo bioluminescence imaging in deep tissues. We injected Gli36-Gluc and Gli36-Rluc cells into the leg muscle of nude mice (n = 4). One week later, mice were injected intravenously with 100 μg (4 mg/kg body weight) of CTZ and imaged immediately using a cooled charge-coupled device (CCD) camera. Four hours later, when signal from the previous imaging session had dropped to background levels, mice were injected with s-CTZ and imaged using the same protocol. Interestingly, the water-soluble CTZ showed 10-fold higher photon counts for Gluc and fourfold for Rluc, as compared with the native CTZ at the same dose, showing that s-CTZ is more sensitive in detecting coelenterate luciferases in vivo (Figure 1a–c). We also determined the effect of increasing the amount of s-CTZ on Gluc and Rluc detection in deep tissues. We injected the same mice mentioned above with 500 μg (20 mg/kg) of water-soluble CTZ and imaged them using a CCD camera. Tumors expressing Gluc resulted in a >120-fold increase in photon counts as compared with tumors imaged with native CTZ (Figure 1a–c). Similarly, Rluc-expressing tumors yielded a 30-fold increase in bioluminescence signal intensity (Figure 1a–c). Importantly, mice did not exhibit any sign of toxicity at this higher dose of s-CTZ. These results collectively demonstrate that the water-soluble CTZ is a sensitive substrate for in vivo imaging of coelenterate luciferase and can be used at a higher dose, overcoming one of the primary limitations of native CTZ, which is its insolubility in water. Recently, we demonstrated that Gluc can be used as a blood reporter for ex vivo monitoring of in vivo biological processes such as gene transfer, viral replication, and circulating cells tracking.15Maguire CA Deliolanis NC Pike L Niers JM Tjon-Kon-Fat LA Sena-Esteves M et al.Gaussia luciferase variant for high-throughput functional screening applications.Anal Chem. 2009; 81: 7102-7106Crossref PubMed Scopus (65) Google Scholar To evaluate the sensitivity of s-CTZ to detect Gluc in the blood, we collected blood samples from mice bearing Gli36-Gluc intramuscular tumors and analyzed 5-μl aliquots (in triplicates) with 100 μl (50 μg/ml) of either CTZ or s-CTZ, using a luminometer. As compared with the native form, the water-soluble CTZ showed a bioluminescence signal more than eightfold higher, demonstrating a greater sensitivity for Gluc detection in the blood (Figure 1d). In summary, we showed that water-soluble CTZ, which is commercially available, is a sensitive substrate for in vivo bioluminescence imaging of coelenterate luciferases. s-CTZ proved to be more sensitive in detecting Gluc and Rluc expression in mammalian cells both in culture and in vivo. Because alcohol is not required, s-CTZ could be injected in mice at higher doses compared with native CTZ, yielding up to 100-fold more light output from tumors expressing Gluc or Rluc. Furthermore, s-CTZ yielded more than an eightfold increase in bioluminescence signal when Gluc was assayed in the blood. Together, these results suggest that s-CTZ is a more potent substrate in detecting subtle coelenterate luciferases in vivo, facilitating noninvasive monitoring of various biological processes. Download .pdf (.04 MB) Help with pdf files Supplementary Methods. Download .pdf (.03 MB) Help with pdf files Supplementary Figure S1. Time kinetics of Gluc and Rluc light production using s-CTZ. Download .pdf (.02 MB) Help with pdf files Supplementary Figure S2. Sensitivity of s-CTZ in detecting coelenterate luciferases in culture. This work was supported by grants from the National Cancer Institute (4R00CA126839) and the National Institute of Neurological Disorders and Stroke (P30NS045776 and 1R01NS064983). We thank Jian Teng and Grant Lewandrowski for technical assistance.

RATIONALE:The role of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) in the development or progression of interstitial lung disease (ILD) is controversial.OBJECTIVES:To evaluate the association between statin use and ILD.METHODS:We used regression analyses to evaluate the association between statin use and interstitial lung abnormalities (ILA) in a large cohort of smokers from COPDGene. Next, we evaluated the effect of statin pretreatment on bleomycin-induced fibrosis in mice and explored the mechanism behind these observations in vitro.MEASUREMENTS AND MAIN RESULTS:In COPDGene, 38% of subjects with ILA were taking statins compared with 27% of subjects without ILA. Statin use was positively associated in ILA (odds ratio, 1.60; 95% confidence interval, 1.03-2.50; P = 0.04) after adjustment for covariates including a history of high cholesterol or coronary artery disease. This association was modified by the hydrophilicity of statin and the age of the subject. Next, we demonstrate that statin administration aggravates lung injury and fibrosis in bleomycin-treated mice. Statin pretreatment enhances caspase-1-mediated immune responses in vivo and in vitro; the latter responses were abolished in bone marrow-derived macrophages isolated from Nlrp3(-/-) and Casp1(-/-) mice. Finally, we provide further insights by demonstrating that statins enhance NLRP3-inflammasome activation by increasing mitochondrial reactive oxygen species generation in macrophages.CONCLUSIONS:Statin use is associated with ILA among smokers in the COPDGene study and enhances bleomycin-induced lung inflammation and fibrosis in the mouse through a mechanism involving enhanced NLRP3-inflammasome activation. Our findings suggest that statins may influence the susceptibility to, or progression of, ILD. Clinical trial registered with www.clinicaltrials.gov (NCT 00608764).

For the analysis of microRNA, a common approach is to first extract microRNA from cellular samples prior to any specific microRNA detection. Thus, it is important to determine the quality and yield of extracted microRNA. In this study, solid-phase extraction was used to isolate small RNA (? Green II staining. Testing for contamination of any small DNA fragments, RNase and cellular peptides or proteins were systematically carried out. By scanning the gel image obtained from PAGE analysis, the average percentage of total microRNA (19 - 25 nt) in the extracted RNA samples was determined to be equal to 2.3 ± 0.5%. The yield of total microRNA was calculated to be ~0.5ng of microRNA per milligram of frozen mouse brain tissue. In comparison to other methods that require the use of expensive specialized instrumentation, the approach of combining the standard UV absorbance and PAGE analysis represents a simple and viable method for evaluating the quality and yield of microRNA extraction from tissue samples.