Plasminogen Binding and Activation at the Mesothelial Cell Surface Promotes Invasion through a Collagen Matrix.

Plasminogen (Plg) activation to the serine protease plasmin (Pla) plays a key role in regulating wound healing and fibrotic responses, particularly when bound to cell surface receptors. Our previous work suggested that mesothelial cells bind Plg at the cell surface, though no Plg receptors were described for these cells. Since mesothelial cells contribute to injury responses, including cellular differentiation to a mesenchymal-like phenotype and extracellular matrix remodeling, we hypothesized that Plg binding would promote these responses. Here, we confirm that Plg binds to both pleural and peritoneal mesothelial cells via the lysine-binding domain present in Plg, and we demonstrate the presence of three Plg receptors on the mesothelial cell surface: α-Enolase, Annexin A2, and Plg-RKT. We further show that bound-Plg is activated to Pla on the cell surface and that activation is blocked by an inhibitor of urokinase plasminogen activator or by the presence of animal-derived FBS. Lastly, we demonstrate that Plg promotes mesothelial cell invasion through a type I collagen matrix but does not promote cellular differentiation or proliferation. These data demonstrate for the first time that mesothelial cells bind and activate Plg at the cell surface and that active Pla is involved in mesothelial cell invasion without cell differentiation.

Late Inflammation Induced by Asbestiform Fibers in Mice Is Ameliorated by a Small Molecule Synthetic Lignan.

Exposure to Libby amphibole (LA) asbestos-like fibers is associated with increased risk of asbestosis, mesothelioma, pulmonary disease, and systemic autoimmune disease. LGM2605 is a small molecule antioxidant and free radical scavenger, with anti-inflammatory effects in various disease models. The current study aimed to determine whether the protective effects of LGM2605 persist during the late inflammatory phase post-LA exposure. Male and female C57BL/6 mice were administered daily LGM2605 (100 mg/kg) via gel cups for 3 days before and 14 days after a 200 µg LA given via intraperitoneal (i.p.) injection. Control mice were given unsupplemented gel cups and an equivalent dose of i.p. saline. On day 14 post-LA treatment, peritoneal lavage was assessed for immune cell influx, cytokine concentrations, oxidative stress biomarkers, and immunoglobulins. During the late inflammatory phase post-LA exposure, we noted an alteration in trafficking of both innate and adaptive immune cells, increased pro-inflammatory cytokine concentrations, induction of immunoglobulin isotype switching, and increased oxidized guanine species. LGM2605 countered these changes similarly among male and female mice, ameliorating late inflammation and altering immune responses in late post-LA exposure. These data support possible efficacy of LGM2605 in the prolonged treatment of LA-associated disease and other inflammatory conditions.

Synthetic secoisolariciresinol diglucoside (LGM2605) inhibits Libby amphibole fiber-induced acute inflammation in mice.

BACKGROUND: Exposure to the Libby amphibole (LA) asbestos-like fibers found in Libby, Montana, is associated with inflammatory responses in mice and humans, and an increased risk of developing mesothelioma, asbestosis, pleural disease, and systemic autoimmune disease. Flaxseed-derived secoisolariciresinol diglucoside (SDG) has anti-inflammatory, anti-fibrotic, and antioxidant properties. We have previously identified potent protective properties of SDG against crocidolite asbestos exposure modeled in mice. The current studies aimed to extend those findings by evaluating the immunomodulatory effects of synthetic SDG (LGM2605) on LA-exposed mice. METHODS: Male and female C57BL/6 mice were given LGM2605 via gavage initiated 3 days prior to and continued for 3 days after a single intraperitoneal dose of LA fibers (200 µg) and evaluated on day 3 for inflammatory cell influx in the peritoneal cavity using flow cytometry. RESULTS: LA exposure induced a significant increase (p < 0.0001) in spleen weight and peritoneal influx of white blood cells, all of which were reduced with LGM2605 with similar trends among males and females. Levels of peritoneal PMN cells were significantly (p < 0.0001) elevated post LA exposure, and were significantly (p < 0.0001) blunted by LGM2605. Importantly, LGM2605 significantly ameliorated the LA-induced mobilization of peritoneal B1a B cells. CONCLUSIONS: LGM2605 reduced LA-induced acute inflammation and WBC trafficking supporting its possible use in mitigating downstream LA fiber-associated diseases. SUMMARY: Following acute exposure to Libby amphibole (LA) asbestos-like fibers, synthetic SDG (LGM2605), a small synthetic molecule, significantly reduced the LA-induced increase in spleen weight and peritoneal inflammation in C57BL/6 male and female mice. Our findings highlight that LGM2605 has immunomodulatory properties and may, thus, likely be a chemopreventive agent for LA-induced diseases.

Analysis of autoantibody profiles in two asbestiform fiber exposure cohorts.

An increased risk for Systemic Autoimmune Diseases (SAID) was reported in the population of Libby, Montana, where extensive exposure to asbestiform amphiboles occurred through mining and use of asbestiform fiber-laden vermiculite. High frequencies of antinuclear autoantibodies (ANA) were detected in individuals and mice exposed to Libby Asbestiform Amphiboles (LAA). Among the 6603 individuals who have undergone health screening at the Center for Asbestos Related Diseases (CARD, Libby MT), the frequencies of rheumatoid arthritis, systemic lupus erythematosus, sarcoidosis, and systemic sclerosis are significantly higher than expected prevalence in the United States. While these data support the hypothesis that LAA can trigger autoimmune responses, evidence suggests that chrysotile asbestos does not. Serological testing was therefore performed in subjects exposed to LAA or predominantly chrysotile (New York steamfitters) using multiplexed array technologies. Analyses were performed in order to determine a) autoantibody profiles in each cohort, and b) whether the two populations could be distinguished through predictive modeling. Analysis using perMANOVA testing confirmed a significant difference between autoantibody profiles suggesting differential pathways leading to autoantibody formation. ANA were more frequent in the LAA cohort. Specific autoantibodies more highly expressed with LAA-exposure were to histone, ribosomal P protein, Sm/Ribonucleoproteins, and Jo-1 (histidyl tRNA synthetase). Myositis autoantibodies more highly expressed in the LAA cohort were Jo-1, PM100, NXP2, and Mi2a. Predictive modeling demonstrated that anti-histone antibodies were most predictive for LAA exposure, and anti-Sm was predictive for the steamfitters' exposure. This emphasizes the need to consider fiber types when evaluating risk of SAID with asbestos exposure.

Autoimmunity and asbestos exposure.

Despite a body of evidence supporting an association between asbestos exposure and autoantibodies indicative of systemic autoimmunity, such as antinuclear antibodies (ANA), a strong epidemiological link has never been made to specific autoimmune diseases. This is in contrast with another silicate dust, crystalline silica, for which there is considerable evidence linking exposure to diseases such as systemic lupus erythematosus, systemic sclerosis, and rheumatoid arthritis. Instead, the asbestos literature is heavily focused on cancer, including mesothelioma and pulmonary carcinoma. Possible contributing factors to the absence of a stronger epidemiological association between asbestos and autoimmune disease include (a) a lack of statistical power due to relatively small or diffuse exposure cohorts, (b) exposure misclassification, (c) latency of clinical disease, (d) mild or subclinical entities that remain undetected or masked by other pathologies, or (e) effects that are specific to certain fiber types, so that analyses on mixed exposures do not reach statistical significance. This review summarizes epidemiological, animal model, and in vitro data related to asbestos exposures and autoimmunity. These combined data help build toward a better understanding of the fiber-associated factors contributing to immune dysfunction that may raise the risk of autoimmunity and the possible contribution to asbestos-related pulmonary disease.

Asbestos-associated mesothelial cell autoantibodies promote collagen deposition in vitro.

Fibrosis, characterized by excessive collagen protein deposition, is a progressive disease that can fatally inhibit organ function. Prolonged exposure to pathogens or environmental toxicants such as asbestos can lead to chronic inflammatory responses associated with fibrosis. Significant exposure to amphibole asbestos has been reported in and around Libby, Montana due to local mining of asbestos-contaminated vermiculite. These exposures have been implicated in a unique disease etiology characterized predominantly by pleural disorders, including fibrosis. We recently reported the discovery of mesothelial cell autoantibodies (MCAAs) in the sera of Libby residents and demonstrated a positive and significant correlation with pleural disease; however, a mechanistic link was not determined. Here we demonstrate that MCAAs induce pleural mesothelial cells to produce a collagen matrix but do not affect production of the pro-inflammatory cytokine tumor growth factor-ß. While autoantibodies commonly induce a pro-fibrotic state by inducing epithelial-mesenchymal transition (EMT) of target cells, we found no evidence supporting EMT in cells exposed to MCAA positive human sera. Although implicated in other models of pulmonary fibrosis, activity of the protein SPARC (secreted protein, acidic and rich in cysteine) did not affect MCAA-induced collagen deposition. However, matrix formation was dependent on matrix metalloproteinase (MMP) activity, and we noted increased expression of MMP-8 and -9 in supernatants of mesothelial cells incubated with MCAA positive sera compared to control. These data suggest a mechanism by which MCAA binding leads to increased collagen deposition through altering MMP expression and provides an important mechanistic link between MCAAs and asbestos-related, autoimmune-induced pleural fibrosis.

Mesothelial cell and anti-nuclear autoantibodies associated with pleural abnormalities in an asbestos exposed population of Libby MT.

Despite data linking amphibole asbestos exposure with production of autoantibodies, the role of autoantibodies in subsequent disease is unknown. Residents of Libby, Montana have experienced significant exposure to amphibole asbestos due to the mining of asbestos-contaminated vermiculite near the community over several decades. This population predominantly exhibits pleural disease, and an autoimmune-like disorder that has yet to be well defined. This study sought to determine whether autoantibodies from asbestos-exposed subjects were associated with pleural lesions. Serum samples of subjects from Libby were evaluated for anti-nuclear antibodies (ANA) and mesothelial cell autoantibodies (MCAA) using cell based ELISA. The presence of radiographic abnormalities detected during the time frame of serum collection was determined from screening records. In accord with previous studies, 61.3% (76/124) of the Libby samples were ANA positive, a frequency much higher than expected for a healthy population. The odds of having pleural or interstitial abnormalities in Libby was nearly 3.55 times greater for individuals that tested positive for ANA compared with individuals negative for ANA (p=0.004). MCAA were also detected at a strikingly high frequency (18.5%; 23/124) in samples from Libby. Individuals with MCAA had 4.9 times the risk of having pleural abnormalities compared to MCAA-negative subjects (p=0.044). In conclusion, ANA and MCAA were elevated in a study population that was known to have chronic exposure to asbestos, and these autoantibodies were associated with pleural abnormalities, the predominant finding in the asbestos-exposed population of Libby. Additional research is needed to determine the role these autoantibodies may play in pulmonary disease.

Validation of an isocratic HPLC method to detect 2-fluoro-beta-alanine for the analysis of dihydropyrimidine dehydrogenase activity.

The efficacy of the chemotherapeutic drug 5'-fluorouracil is reduced by catabolism to 2'-fluoro-beta-alanine (FBAL), a three-step reaction in which dihydropyrimidine dehydrogenase (DPD) catalyzes the rate-limiting step. To study in vitro DPD activity, we developed and validated an isocratic, reverse-phase HPLC method to detect and quantify FBAL without using multiple columns or radiolabeled substrates. Pre-column derivatization of FBAL was performed using o-phthalaldehyde in the presence of two sulfur donors, ethanthiol or beta-mercaptoethanol, and the resulting products assayed. Calibration curves were linear over a range of 10-200 microg/ml and the method was successfully applied to the examination of DPD activity in cultured cells.

Development and validation of a rapid and sensitive HPLC method for the quantification of 5-fluorocytosine and its metabolites.

To study the intracellular metabolism of the prodrug 5-fluorocytosine (5FC), we developed a novel reverse-phase high-performance liquid chromatography method to simultaneously detect 5FC and its four major anabolic metabolites: 5-fluorouracil, 5-fluorouridine, 5-fluorouridine-monophosphate and 5-fluoro-2'deoxyuridine-5'-monophosphate. Separation of each compound was accomplished under isocratic conditions using a C(18) column and mobile phase of formic acid-water (1 : 99 v/v). The method was validated for both accuracy and reproducibility in cell culture media. Additionally, metabolites were assessed for stability at ambient temperatures and following freeze-thaw cycles. Calibration curves were linear over a range of 1-200 microg/mL. Limit of quantification for four of the five compounds was 1 microg/mL in cell culture media (RSD < 11%). This method was successfully used to monitor intracellular conversion of 5FC to its metabolic products over a 24h period.