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

Abstract

Multi-walled 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 (TGFβ) 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, TGFβ, 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 extravation. LPS and MWCNT increased the expression of TGFβ 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.

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

“…In lung fibroblasts, MWCNT induce the expression of TGFβ receptor 1 (TGFβR1), TGFβ1, SMAD2/3, and collagen synthesis . In alveolar epithelial cells, MWCNT induce the expression of ECM components TGFβ and Col3A1, and remodeling zinc‐metalloproteinases MMP‐9 and MMP‐12 . In support of the in vitro studies, the in vivo studies also showed the production of TGFβ and MMP‐9 in bronchoalveolar lavage cells (BAL) or whole lung lysate of mice treated with MWCNT .…”

“…Numerous studies previously have shown that MWCNT induce lung fibrosis in experimental animal models . The fibrotic potential of MWCNT is mediated by the activation of TGFβ signaling pathway . Mishra et al showed that MWCNT activate TGFβ signaling pathway by upregulating the expression of its receptor, TGFβ R1, and downstream activation of Smad2/3 proteins.…”

“…MWCNT were purchased from Sigma‐Aldrich (St. Louis, MO) and characterized as previously described in Pacurari et al…”

“…Wound healing assay was performed according to the methods previously described . A549 cells were grown on cover slips to a confluent monolayer and then scratched to form a 100‐μm “wound” using 100 µL sterile pipette tips.…”

“…Our previous study showed that MWCNT activate alveolar epithelial A549 cells to express fibrosis markers TGFβ and Col 3A1. MWCNT induced TGFβ by 2.62‐, and Co3A1 by 2.34‐fold change versus control . Although, several studies have shown that MWCNT induce molecular factors associated with lung toxicity, the exact molecular factors involved in MWCNT signaling still remain unknown.…”