MIP-4 is Induced by Bleomycin and Stimulates Cell Migration Partially via Nir-1 Receptor.

Background: CC-chemokine ligand 18 also known as MIP-4 is a chemokine with roles in inflammation and immune responses. It has been shown that MIP-4 is involved in the development of several diseases including lung fibrosis and cancer. How exactly MIP-4 is regulated and exerts its role in lung fibrosis remains unclear. Therefore, in the present study, we examined how MIP-4 is regulated and whether it acts via its potential receptor Nir-1. Materials and Methods: A549 cells were grown and maintained in DMEM : F12 (1 : 1) and supplemented with 10% FBS and 1000 U of penicillin/streptomycin and maintained as recommended by the manufacturer (ATCC). Cell migration and invasion, immunohistochemistry (IHC), Western blot, qPCR, and siRNA Nir-1 were used to determine MIP-4 regulation and its role in cell migration. Results: Cell migration was increased following stimulation of cells with recombinant (r) MIP-4 and bleomycin (BLM), whereas quenching rMIP-4 with its antibody (Ab) or addition of the Ab to BLM or H2O2 diminished rMIP-4-induced cell migration. Along with cell migration, rMIP-4, BLM, and H2O2 induced the formation of actin filaments dynamic structures whereas costimulation with MIP-4 Ab limited BLM- and H2O2-induced effects. MIP-4 mRNA and protein were increased by BLM and H2O2, and the addition of its Ab significantly reduced treatments effect. Experiments with siRNA investigating whether Nir-1 is a potential MIR-4 receptor indicated that the inhibition of Nir-1 decreased cell migration/invasion but did not totally inhibit rMIP-4-induced cell migration. Conclusion: Therefore, our data indicate that MIP-4 is regulated by BLM and H2O2 and costimulation with its Ab limits the effects on MIP-4 and that the Nir-1 receptor partially mediates MIP-4's effects on increased cell migration. These data also evidenced that MIP-4 is regulated by fibrotic and oxidative stimuli and that quenching MIP-4 with its Ab or therapeutically targeting the Nir-1 receptor may partially limit MIP-4 effects under fibrotic or oxidative stimulation.

Acute Vibration Induces Peripheral Nerve Sensitization in a Rat Tail Model: Possible Role of Oxidative Stress and Inflammation.

Prolonged occupational exposure to hand-held vibrating tools leads to pain and reductions in tactile sensitivity, grip strength and manual dexterity. The goal of the current study was to use a rat-tail vibration model to determine how vibration frequency influences factors related to nerve injury and dysfunction. Rats were exposed to restraint, or restraint plus tail vibration at 62.5 Hz or 250 Hz. Nerve function was assessed using the current perception threshold (CPT) test. Exposure to vibration at 62.5 and 250 Hz, resulted in a reduction in the CPT at 2000 and 250-Hz electrical stimulation (i.e. increased Aß and Aδ, nerve fiber sensitivity). Vibration exposure at 250 Hz also resulted in an increased sensitivity of C-fibers to electrical stimulation and thermal nociception. These changes in nerve fiber sensitivity were associated with increased expression of interleukin (IL)-1ß and tumor necrosis factor (TNF)-α in ventral tail nerves, and increases in circulating concentrations of IL-1 ß in rats exposed to 250-Hz vibration. There was an increase in glutathione, but no changes in other measures of oxidative activity in the peripheral nerve. However, measures of oxidative stress were increased in the dorsal root ganglia (DRG). These changes in pro-inflammatory factors and markers of oxidative stress in the peripheral nerve and DRG were associated with inflammation, and reductions in myelin basic protein and post-synaptic density protein (PSD)-95 gene expression, suggesting that vibration-induced changes in sensory function may be the result of changes at the exposed nerve, the DRG and/or the spinal cord.

Thrombospondin-1 and microRNA-1 expression in response to multiwalled carbon nanotubes in alveolar epithelial cells.

Thrombospondin-1 (TSP-1) is a glycoprotein that plays a role in extracellular matrix (ECM) remodeling. Previously, we have shown that multiwalled carbon nanotubes (MWCNT) regulate ECM components TGFß and its target Col3A1 in alveolar epithelial cells. In this study, we investigated the effect of MWCNT on TSP-1 and microRNA-1 (miR-1) in the regulation of TGFß in ECM remodeling using alveolar epithelial A549 cells. A549 cells were treated with MWCNT (20 or 50 µg/mL) for 6 or 24 h and the expression of TSP-1 and miR-1, and the exogenous miR-1 effect on cell morphology were analyzed. MWCNT induced in a time- and dose-dependent manner the expression of TSP-1. miR-1 was suppressed by MWCNT after 6 or 24 h of treatment regardless of the dose. TSP-1 and miR-1 negatively correlated with each other, r = -0.58. Exogenous administration of miR-1 induced alveolar epithelial cell morphology changes including cell clustering, whereas inhibition of miR-1 induced less cell to cell contact, cell rounding, and cellular projections. IntAct molecular network interactions analysis revealed that TSP-1 interacts with 21 molecular factors including ECM genes, and molecules. These results indicate a relationship between that TSP-1, MWCNT, and TGFß, and suggest TSP-1 may play a role in MWCNT-induced TGFß and ECM remodeling. Moreover, these data also suggest an inverse relationship between TSP-1 and miR-1 and a potential role of miR-1 in MWCNT-induced fibrotic signaling. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1596-1606, 2017.

Effects of lipopolysaccharide, multiwalled carbon nantoubes, and the combination on lung alveolar epithelial cells.

Multiwalled carbon nanotubes (MWCNT) have been shown to induce lung fibrosis in animal models, however the underlying molecular factors/mechanisms are still unclear. In this study, we investigated the effects of lipopolysaccharide (LPS), MWCNT, and the combination of LPS and MWCNT on the expression of matrix metalloproteinase-9 and metalloproteinase-12 (MMP-9, MMP-12), collagen 3A1 (Col3A1), and transforming growth factor beta (TGFß) in alveolar epithelial A549 cells. MMPs are proteinases that degrade extracellular matrix and play a role in lung fibrosis. A549 cells were exposed to LPS (1 ng/mL), MWCNT (20 µg/mL), and the combination and analyzed for paracellular permeability, TGFß, Col3A1, MMP-9, MMP-12, NF-κB activation, and cell migration by real-time PCR and immunofluorescence. LPS, the combination of LPS and MWCNT, and MWCNT only at the highest tested dose induced blue dextran extravasation. LPS and MWCNT increased the expression of TGFß and its downstream target gene Col3A, and MMP-9 and MMP-12 mRNA. MWCNT potently induced cell migration toward wound healing, whereas LPS slightly induced cell migration. Both, LPS and MWCNT, induced NF-κB nuclear translocation. Our results indicate that MWCNT activated alveolar epithelial cells to promote fibrogenesis, and that LPS differentially primes molecular factors involved in lung remodeling. These findings suggest a role of alveolar epithelial cells in fibrogenesis and also may aid in the design and development of tests for screening of fibrogenic agents. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 445-455, 2017.

Cell permeability, migration, and reactive oxygen species induced by multiwalled carbon nanotubes in human microvascular endothelial cells.

Multiwalled carbon nanotubes (MWCNT) have elicited great interest in biomedical applications due to their extraordinary physical, chemical, and optical properties. Intravenous administration of MWCNT-based medical imaging agents and drugs in animal models was utilized. However, the potential harmful health effects of MWCNT administration in humans have not yet been elucidated. Furthermore, to date, there are no apparent reports regarding the precise mechanisms of translocation of MWCNT into target tissues and organs from blood circulation. This study demonstrates that exposure to MWCNT leads to an increase in cell permeability in human microvascular endothelial cells (HMVEC). The results obtained from this study also showed that the MWCNT-induced rise in endothelial permeability is mediated by reactive oxygen species (ROS) production and actin filament remodeling. In addition, it was found that MWCNT promoted cell migration in HMVEC. Mechanistically, MWCNT exposure elevated the levels of monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule 1 (ICAM-1) in HMVEC. Taken together, these results provide new insights into the bioreactivity of MWCNT, which may have implications in the biomedical application of MWCNT in vascular targeting, imaging, and drug delivery. The results generated from this study also elucidate the potential adverse effects of MWCNT exposure on humans at the cellular level.

Cell permeability, migration, and reactive oxygen species induced by multiwalled carbon nanotubes in human microvascular endothelial cells.

Multiwalled carbon nanotubes (MWCNT) have elicited great interest in biomedical applications due to their extraordinary physical, chemical, and optical properties. Intravenous administration of MWCNT-based medical imaging agents and drugs in animal models was utilized. However, the potential harmful health effects of MWCNT administration in humans have not yet been elucidated. Furthermore, to date, there are no apparent reports regarding the precise mechanisms of translocation of MWCNT into target tissues and organs from blood circulation. This study demonstrates that exposure to MWCNT leads to an increase in cell permeability in human microvascular endothelial cells (HMVEC). The results obtained from this study also showed that the MWCNT-induced rise in endothelial permeability is mediated by reactive oxygen species (ROS) production and actin filament remodeling. In addition, it was found that MWCNT promoted cell migration in HMVEC. Mechanistically, MWCNT exposure elevated the levels of monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule 1 (ICAM-1) in HMVEC. Taken together, these results provide new insights into the bioreactivity of MWCNT, which may have implications in the biomedical application of MWCNT in vascular targeting, imaging, and drug delivery. The results generated from this study also elucidate the potential adverse effects of MWCNT exposure on humans at the cellular level.

Multi-walled carbon nanotube-induced gene expression in the mouse lung: association with lung pathology.

Due to the fibrous shape and durability of multi-walled carbon nanotubes (MWCNT), concerns regarding their potential for producing environmental and human health risks, including carcinogenesis, have been raised. This study sought to investigate how previously identified lung cancer prognostic biomarkers and the related cancer signaling pathways are affected in the mouse lung following pharyngeal aspiration of well-dispersed MWCNT. A total of 63 identified lung cancer prognostic biomarker genes and major signaling biomarker genes were analyzed in mouse lungs (n=80) exposed to 0, 10, 20, 40, or 80µg of MWCNT by pharyngeal aspiration at 7 and 56days post-exposure using quantitative PCR assays. At 7 and 56days post-exposure, a set of 7 genes and a set of 11 genes, respectively, showed differential expression in the lungs of mice exposed to MWCNT vs. the control group. Additionally, these significant genes could separate the control group from the treated group over the time series in a hierarchical gene clustering analysis. Furthermore, 4 genes from these two sets of significant genes, coiled-coil domain containing-99 (Ccdc99), muscle segment homeobox gene-2 (Msx2), nitric oxide synthase-2 (Nos2), and wingless-type inhibitory factor-1 (Wif1), showed significant mRNA expression perturbations at both time points. It was also found that the expression changes of these 4 overlapping genes at 7days post-exposure were attenuated at 56days post-exposure. Ingenuity Pathway Analysis (IPA) found that several carcinogenic-related signaling pathways and carcinogenesis itself were associated with both the 7 and 11 gene signatures. Taken together, this study identifies that MWCNT exposure affects a subset of lung cancer biomarkers in mouse lungs.

Estrogen potentiates the combined effects of transforming growth factor-beta and tumor necrosis factor-alpha on adult human osteoblast-like cell prostaglandin E2 biosynthesis.

Reports that estrogen treatment modulates arachidonic acid metabolism by bone and bone cells are found in the literature. However, conflicting indications of the relationship that exists between estrogen and arachidonic acid metabolism emerge from the analysis of those reports. The present studies were undertaken to determine if estrogen effected the production of prostaglandins (PG) in human osteoblast-like (hOB) cell cultures derived from adults, under basal or cytokine-stimulated conditions. A 48-hour estrogen pretreatment did not modify hOB cell PG biosynthesis on a qualitative basis, and PGE2 formation predominated under all tested conditions. Estrogen pretreatment did lead to increased PGE2 production in specimens stimulated conjointly with transforming growth factor-beta1 and tumor necrosis factor-alpha ( p < 0.001). No changes in PGE2 production were observed in estrogen pretreated specimens stimulated singly with either tested cytokine, nor in samples in which either TGFbeta or TNF was replaced by interleukin-1beta. Anti-estrogen (ICI 164,384) inclusion prevented the estrogen-dependent increase in PGE2 production in the TGFbeta plus TNF-stimulated samples. These results suggest that an estrogen effect on bone cell prostaglandin biosynthesis may be most evident and significant under conditions in which the cells are exposed to multiple osteotropic cytokines, a condition that applies during the bone remodeling process.