David J. Blake

Targeting Nrf2 with the triterpenoid CDDO-imidazolide attenuates cigarette smoke-induced emphysema and cardiac dysfunction in mice

Chronic obstructive pulmonary disease (COPD), which comprises emphysema and chronic bronchitis resulting from prolonged exposure to cigarette smoke (CS), is a major public health burden with no effective treatment. Emphysema is also associated with pulmonary hypertension, which can progress to right ventricular failure, an important cause of morbidity and mortality among patients with COPD. Nuclear erythroid 2 p45 related factor-2 (Nrf2) is a redox-sensitive transcription factor that up-regulates a battery of antioxidative genes and cytoprotective enzymes that constitute the defense against oxidative stress. Recently, it has been shown that patients with advanced COPD have a decline in expression of the Nrf2 pathway in lungs, suggesting that loss of this antioxidative protective response is a key factor in the pathophysiological progression of emphysema. Furthermore, genetic disruption of Nrf2 in mice causes early-onset and severe emphysema. The present study evaluated whether the strategy of activation of Nrf2 and its downstream network of cytoprotective genes with a small molecule would attenuate CS-induced oxidative stress and emphysema. Nrf2+/+ and Nrf2−/− mice were fed a diet containing the potent Nrf2 activator, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), while being exposed to CS for 6 months. CDDO-Im significantly reduced lung oxidative stress, alveolar cell apoptosis, alveolar destruction, and pulmonary hypertension in Nrf2+/+ mice caused by chronic exposure to CS. This protection from CS-induced emphysema depended on Nrf2, as Nrf2−/− mice failed to show significant reduction in alveolar cell apoptosis and alveolar destruction after treatment with CDDO-Im. These results suggest that targeting the Nrf2 pathway during the etiopathogenesis of emphysema may represent an important approach for prophylaxis against COPD.

Deletion of Keap1 in the lung attenuates acute cigarette smoke-induced oxidative stress and inflammation.

Exposure to cigarette smoke (CS) is the primary factor associated with the development of chronic obstructive pulmonary disease (COPD). CS increases the level of oxidants in the lungs, resulting in a depletion of antioxidants, which promotes oxidative stress and the destruction of alveolar tissue. In response to CS, pulmonary epithelial cells counteract increased levels of oxidants by activating Nrf2-dependent pathways to augment the expression of detoxification and antioxidant enzymes, thereby protecting the lung from injury. We hypothesize that increasing the pathways activated by Nrf2 will afford protection against CS-induced lung damage. To this end we have developed a novel mouse model in which the cytosolic inhibitor of Nrf2, Keap1, is genetically deleted in Clara cells, which predominate in the upper airways in mice. Deletion of Keap1 in Clara cells resulted in increased expression of Nrf2-dependent genes, such as Nqo1 and Gclm, as determined by microarray analysis and quantitative PCR. Deletion of Keap1 in airway epithelium decreased Keap1 protein levels and significantly increased the total level of glutathione in the lungs. Increased Nrf2 activation protected Clara cells against oxidative stress ex vivo and attenuated oxidative stress and CS-induced inflammation in vivo. Expression of KEAP1 was also decreased in human epithelial cells through siRNA transfection, which increased the expression of Nrf2-dependent genes and attenuated oxidative stress. In conclusion, activating Nrf2 pathways in tissue-specific Keap1 knockout mice represents an important genetic approach against oxidant-induced lung damage.

The effect of volume on the distribution of substances instilled into the peritoneal cavity

Abstract 99m Tc-labeled human serum albumin was placed into the peritoneal cavity of monkeys with 20- and 250-ml volumes of vehicle. Its distribution was monitored with a γ camera. Only the large volume injections produced uniform distribution of the label.

Asbestos-Induced Autoimmunity in C57Bl/6 Mice

Environmental impacts on autoimmunity have significant public health implications. Epidemiological studies have shown associations between exposure to airborne silicates, such as crystalline silica or asbestos, and autoimmunity, but the etiology remains unclear. The purpose of this study was to test the hypothesis that asbestos could lead to a specific pattern of autoantibodies and pathology indicative of systemic autoimmune disease (SAID). Female C57Bl/6 mice were instilled intratracheally with 2 doses × 60 μ g/mouse of amphibole asbestos (tremolite), wollastonite (a non-fibrogenic control fiber), or saline alone. Serum samples were collected and urine was checked for protein bi-weekly for 7 months. By 26 weeks, the asbestos-instilled animals had a significantly higher frequency of positive anti-nuclear antibody (ANA) tests compared to wollastonite and saline groups. The majority of positive ANAs showed homogeneous or combined homogeneous/speckled patterns, and tested positive for antibodies to dsDNA and SSA/Ro 52. Serum isotyping showed no significant changes in IgM, IgA, or IgG subclasses. However, there was an overall decrease in the mean IgG serum concentration in asbestos-instilled mice. IgG immune complex deposition was demonstrated in the kidneys of asbestos-instilled mice, with evidence of glomerular and tubule abnormalities suggestive of glomerulonephritis. Flow cytometry demonstrated moderate changes in the percentages of CD25+ T-suppressor cells and B1a B-cells in the superficial cervical lymph nodes of the asbestos-instilled mice. These data demonstrate that asbestos leads to immunologic changes consistent with the development of autoimmunity. This study provides a non-autoimmune prone murine model for use in future elucidation of mechanisms involved in asbestos-induced autoimmune disease.

Sulforaphane inhibits de novo synthesis of IL-8 and MCP-1 in human epithelial cells generated by cigarette smoke extract

Chronic obstructive pulmonary disease (COPD) is currently the fifth leading cause of death worldwide. Exposure to cigarette smoke (CS) is the primary factor associated with the COPD development. CS activates epithelial cells to secrete chemokines such as interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) that recruit neutrophils and macrophages to the lung. These inflammatory cells then release additional chemokines and cytokines leading to chronic inflammation that initiates apoptosis in epithelial and endothelial cells and destruction of alveolar structure. Pulmonary epithelium responds to oxidative stress mediated by CS through activating NRF2-dependent pathways, leading to an increased expression of antioxidant and cytoprotective enzymes thereby providing a protective response against CS-induced lung injury. We hypothesized that activating NRF2-dependent cytoprotective gene expression with sulforaphane (SFN) affords protection against CS-induced lung damage by inhibiting chemokine production. Results indicate that in the human BEAS-2B epithelial cell line, 5 μM SFN activated NRF2-dependent gene expression by triggering the translocation of NRF2 to the nucleus and significantly increased the expression of NRF2-dependent genes such as NADPH quinone oxidoreductase-1, heme oxygenase-1, and glutamate cysteine ligase modulatory subunit. Cigarette smoke extract (CSE) exposure of BEAS-2B cells significantly increased production of both IL-8 and MCP-1. Production of both chemokines was significantly reduced with SFN given prior to CSE; SFN inhibited IL-8 and MCP-1 gene expression at the transcription level. Our results indicate that activating NRF2 pathways with SFN inhibits CSE-induced chemokine production in human epithelial cells. However, the mechanism by which the production of chemokines is inhibited through SFN still remains to be elucidated. SFN may enhance NRF2 transcriptional activity resulting in the inhibition of proinflammatory pathways such as NF-κB.

Autoantibodies from mice exposed to Libby amphibole asbestos bind SSA/Ro52-enriched apoptotic blebs of murine macrophages.

Asbestos exposure is associated with increased autoimmune responses in humans. For example, in Libby, MT where significant asbestos exposure has occurred due to an asbestos-contaminated vermiculite mine near the community, residents have developed increased autoimmune responses compared to an unexposed population. However, the exact mechanism by which Libby amphibole asbestos generates autoimmune responses is unclear. A murine model of amphibole asbestos-induced autoimmunity was recently established, and one of the targets of the autoantibodies (AAs) was the SSA/Ro52 autoantigen. The purpose of this study was to determine whether the SSA/Ro52 autoantigen is exposed at the surface of cells as a result of asbestos exposure as a possible mechanism leading to antigenicity. Our results indicate that Libby asbestos induces apoptosis in murine macrophages as determined by phosphatidylserine exposure, cleavage of poly(ADP-ribose) polymerase and morphological changes such as nuclear condensation. Moreover, asbestos-induced apoptosis results in the formation of apoptotic cell surface blebs enriched in SSA/Ro52 as determined by confocal microscopy. Most importantly, apoptotic cell surface blebs are recognized by AAs from mice exposed to amphibole asbestos suggesting that these cell surface structures may be antigenic when presented in a pro-inflammatory context. This study supports the hypothesis that the induction of apoptosis plays a key role in environmentally induced autoimmunity through cell surface exposure of a known autoantigen.

Soluble extracellular Klotho decreases sensitivity to cigarette smoke induced cell death in human lung epithelial cells

Chronic obstructive pulmonary disease (COPD) is currently the third leading cause of death in the US and is associated with an abnormal inflammatory response to cigarette smoke (CS). Exposure to CS induces oxidative stress and can result in cellular senescence in the lung. Cellular senescence can then lead to decreased proliferation of epithelial cells, the destruction of alveolar structure and pulmonary emphysema. The anti-aging gene, klotho, encodes a membrane bound protein that has been shown to be a key regulator of oxidative stress and cellular senescence. In this study the role of Klotho (KL) with regard to oxidative stress and cellular senescence was investigated in human pulmonary epithelial cells exposed to cigarette smoke. Individual clones that stably overexpress Klotho were generated through retroviral transfection and geneticin selection. Klotho overexpression was confirmed through RT-qPCR, Western blotting and ELISA. Compared to control cells, constitutive Klotho overexpression resulted in decreased sensitivity to cigarette smoke induced cell death in vitro via a reduction of reactive oxygen species and a decrease in the expression of p21. Our results suggest that increasing Klotho level in pulmonary epithelial cells may be a promising strategy to reduce cellular senescence and mitigate the risk for the development of COPD.

Antiviral activity of (+)-sattabacin against varicella zoster.

The first report of the antiviral activity of (+)-sattabacin against varicella-zoster virus (VZV) is described. Our results show that (+)-sattabacin potently inhibits the growth of VZV at concentrations in the range of other drugs commonly prescribed for VZV infection. Experiments detailing the synthesis of (+)-sattabacin, quantification of cytotoxicity and gene expression data in human fibroblast cells are also presented. Gene expression data was obtained through microarray analysis from human fibroblast cells exposed to sattabacin in order to identify a possible mechanism by which (+)-sattabacin inhibits VZV replication.

4-Methyl-3-nitro-benzoic acid, a migration inhibitor, prevents breast cancer metastasis in SCID mice

Metastasis remains a formidable problem in malignant tumors. In this study, MTT assay revealed that 4-methyl-3-nitro-benzoic acid (MNBA) had no effect on cell viability and did not interfere with cell cycle in any breast cancer cell lines tested. However, treatment with MNBA on breast cancer cells can inhibit EGF-induced migration and chemotaxis in vitro. In vivo assay demonstrated that MNBA and Paclitaxel synergistically inhibited tumor growth and metastasis in breast cancer SCID mice xenografts. These results suggest that MNBA is a potent inhibitor cancer cell chemotaxis and may be developed into a novel anti-metastasis drug.

Ablation of the CD9 receptor in human lung cancer cells using CRISPR/Cas alters migration to chemoattractants including IL-16

Graphical abstract Figure. No caption available. HighlightsCD9 expression was ablated in A549 cells using CRISPR/Cas technology.Wild‐type A549 cells migrated towards chemoattractants, however, CD9 expression was required in this process.CD9 knockdown cells had a significantly reduced migration towards growth serum and IL‐16.IL‐16 stimulation does not lead to the activation of a stress response in A549 cells. Abstract CD9, a member of the tetraspanin superfamily, has been implicated in regulating various physiological processes, including cell motility, adhesion, apoptosis and metastasis. Recently, interleukin‐16 (IL‐16), a pro‐inflammatory cytokine released by normal airway and alveolar epithelial cells, has been implicated as a possible ligand for CD9 as an alternative receptor. In this study, using A549 cells as a model of human alveolar epithelium, CD9 expression was ablated using CRISPR/Cas technology. Decreased expression of CD9 mRNA and protein levels was confirmed through RT‐qPCR and flow cytometry, respectively. Individual clones were generated that expressed high levels of CD9 (wild‐type, WT) or significantly less CD9 (knockdown, KD). Both wild‐type and knockdown A549 cell migration was quantified using a FluoroBloc transwell chemotaxis assay. Our results indicate that wild‐type A549 cells migrated towards chemoattractants. Moreover, CD9 expression was required in this process since the CD9 knockdown cells had a significantly reduced migration towards growth serum and IL‐16. These results support the migratory properties for CD9 in human lung cells and support the hypothesis that CD9 serves as an alternative receptor for IL‐16.