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Nature communications  2014;5:4753.
Circadian rhythms are known to regulate immune responses in healthy animals, but it is unclear whether they persist during acute illnesses where clock gene expression is disrupted by systemic inflammation. Here, we use a genome-wide approach to investigate circadian gene and metabolite expression in the lungs of endotoxemic mice and find that novel cellular and molecular circadian rhythms are elicited in this setting. The endotoxin-specific circadian program exhibits unique features, including a divergent group of rhythmic genes and metabolites compared to the basal state and a distinct periodicity and phase distribution. At the cellular level endotoxin treatment also alters circadian rhythms of leukocyte counts within the lung in a bmal1-dependent manner, such that granulocytes rather than lymphocytes become the dominant oscillating cell type. Our results show that inflammation produces a complex reorganization of cellular and molecular circadian rhythms that are relevant to early events in lung injury.
PMCID: PMC4162491  PMID: 25208554
2.  The Inflammasome Mediates Hyperoxia-Induced Alveolar Cell Permeability 
A hallmark of hyperoxic acute lung injury is the influx of inflammatory cells to lung tissue and the production of proinflammatory cytokines, such as IL-1β; however, the mechanisms connecting hyperoxia and the inflammatory response to lung damage is not clear. The inflammasome protein complex activates caspase-1 to promote the processing and secretion of proinflammatory cytokines. We hypothesized that hyperoxia-induced K+ efflux activates the inflammasome via the purinergic P2X7 receptor to cause inflammation and hyperoxic acute lung injury. To test this hypothesis, we characterized the expression and activation of inflammasome components in primary murine alveolar macrophages exposed to hyperoxia (95% oxygen and 5% CO2) in vitro, and in alveolar macrophages isolated from mice exposed to hyperoxia (100% oxygen). Our results showed that hyperoxia increased K+ efflux, inflammasome formation, release of proinflammatory cytokines, and induction of caspase-1 and IL-1β cleavage both in vitro and in vivo. The P2X7 agonist ATP enhanced hyperoxia-induced inflammasome activation, whereas the P2X7 antagonist, oxidized ATP, inhibited hyperoxia induced inflammasome activation. In addition, when ATP was scavenged with apyrase, hyperoxia-induced inflammasome activation was significantly decreased. Furthermore, short hairpin RNA silencing of inflammasome components abrogated hyperoxia-induced secretion of proinflammatory cytokines in vitro. These results suggest that hyperoxia induces K+ efflux through the P2X7 receptor, leading to inflammasome activation and secretion of proinflammatory cytokines. These events would affect the permeability of the alveolar epithelium and ultimately lead to epithelial barrier dysfunction and cell death.
PMCID: PMC3780794  PMID: 20375306
3.  MicroRNA-21 Integrates Pathogenic Signaling to Control Pulmonary Hypertension: Results of a Network Bioinformatics Approach 
Circulation  2012;125(12):1520-1532.
Pulmonary hypertension (PH) is driven by diverse pathogenic etiologies. Owing to their pleiotropic actions, microRNA (miRNA) are potential candidates for coordinated regulation of these disease stimuli.
Methods and Results
Using a network biology approach, we identify miRNA associated with multiple pathogenic pathways central to PH. Specifically, microRNA-21 (miR-21) is predicted as a PH-modifying miRNA, regulating targets integral to bone morphogenetic protein (BMP) and Rho/Rho kinase signaling as well as functional pathways associated with hypoxia, inflammation, and genetic haplo insufficiency of the BMP Receptor Type 2 (BMPRII). To validate these predictions, we have found that hypoxia and BMPRII signaling independently up-regulate miR-21 in cultured pulmonary arterial endothelial cells. In a reciprocal feedback loop, miR-21 down-regulates BMPRII expression. Furthermore, miR-21 directly represses RhoB expression and Rho kinase activity, inducing molecular changes consistent with decreased angiogenesis and vasodilation. In vivo, miR-21 is up-regulated in pulmonary tissue from several rodent models of PH and in humans with PH. Upon induction of disease in miR-21-null mice, RhoB expression and Rho-kinase activity are increased, accompanied by exaggerated manifestations of PH.
A network-based bioinformatic approach coupled with confirmatory in vivo data delineates a central regulatory role for miR-21 in PH. Furthermore, this study highlights the unique utility of network biology for identifying disease-modifying miRNA in PH.
PMCID: PMC3353408  PMID: 22371328
Pulmonary Heart Disease; microRNA; Network Biology; Molecular Biology; Vasculature
4.  Characterization of macroautophagic flux in vivo using a leupeptin-based assay 
Autophagy  2011;7(6):629-642.
Macroautophagy is a highly conserved catabolic process that is crucial for organ homeostasis in mammals. However, methods to directly measure macroautophagic activity (or flux) in vivo are limited. In this study we developed a quantitative macroautophagic flux assay based on measuring LC3b protein turnover in vivo after administering the protease inhibitor leupeptin. Using this assay we then characterized basal macroautophagic flux in different mouse organs. We found that the rate of LC3b accumulation after leupeptin treatment was greatest in the liver and lowest in spleen. Interestingly we found that LC3a, an ATG8/LC3b homologue and the LC3b-interacting protein p62 were degraded with similar kinetics to LC3b. However, the LC3b-related proteins GABARAP and GATE-16 were not rapidly turned over in mouse liver, implying that different LC3b homologues may contribute to macroautophagy via distinct mechanisms. Nutrient starvation augmented macroautophagic flux as measured by our assay, while refeeding the animals after a period of starvation significantly suppressed flux. We also confirmed that beclin 1 heterozygous mice had reduced basal macroautophagic flux compared to wild-type littermates. These results illustrate the usefulness of our leupeptin-based assay for studying the dynamics of macroautophagy in mice.
PMCID: PMC3127049  PMID: 21460622
macroautophagy; autophagy; flux; mice; in vivo; LC3; GABARAP; GATE-16; leupeptin; cycloheximide
5.  Non-invasive assessment of murine pulmonary arterial pressure: validation and application to models of pulmonary hypertension 
Genetically-modified mice offer the unique opportunity to gain insights into the pathophysiology of pulmonary arterial hypertension (PAH). In mice, right heart catheterization is the only available technique to measure right ventricular systolic pressure (RVSP). However, it is a terminal procedure and does not allow for serial measurements. Our objective was to validate a non-invasive technique to assess RVSP in mice.
Methods and Results
Right ventricle catheterization and echocardiography (30-MHz transducer) were simultaneously performed in mice with pulmonary hypertension induced acutely by infusion of a thromboxane analogue, U-46619, or chronically by lung-specific over-expression of interleukin 6 (IL-6). Pulmonary acceleration time (PAT) and ejection time (ET) were measured in the parasternal short axis view by pulsed-wave Doppler of pulmonary artery flow. Infusion of U-46619 acutely increased RVSP, shortened PAT, and decreased PAT/ET. The pulmonary flow pattern changed from symmetric at baseline to asymmetric at higher RVSPs. In wild-type and IL-6-over-expressing mice, the PAT correlated linearly with RVSP (r2=−0.67; p<0.0001), as did PAT/ET (r2=−0.76; p<0.0001). Sensitivity and specificity for detecting high RVSP (>32 mmHg) were 100% (7/7) and 86% (6/7), respectively, for both indices (cutoff values: PAT <21 ms and PAT/ET <39%). Intra-observer and inter-observer variability of PAT and PAT/ET were less than 6%.
Right ventricular systolic pressure can be estimated non-invasively in mice. Echocardiography is able to detect acute and chronic increases in RVSP with high sensitivity and specificity, as well as to assess the effects of treatment on RVSP. This non-invasive technique may permit the characterization of the evolution of PAH in genetically-modified mice.
PMCID: PMC3075498  PMID: 20044514
Echocardiography; right ventricular systolic pressure; mice

Results 1-5 (5)