SOX2Mediates Carbon Nanotube-Induced Fibrogenesis and Fibroblast Stem Cell Acquisition.

Certain nanosized particles like carbon nanotubes (CNTs) are known to induce pulmonary fibrosis, but the underlying mechanisms are unclear, and efforts to prevent this disease are lacking. Fibroblast-associated stem cells (FSCs) have been suggested as a critical driver of fibrosis induced by CNTs by serving as a renewable source of extracellular matrix-producing cells; however, a detailed understanding of this process remains obscure. Here, we demonstrated that single-walled CNTs induced FSC acquisition and fibrogenic responses in primary human lung fibroblasts. This was indicated by increased expression of stem cell markers (e.g., CD44 and ABCG2) and fibrogenic markers (e.g., collagen and α-SMA) in CNT-exposed cells. These cells also showed increased sphere formation, anoikis resistance, and aldehyde dehydrogenase (ALDH) activities, which are characteristics of stem cells. Mechanistic studies revealed sex-determining region Y-box 2 (SOX2), a self-renewal associated transcription factor, as a key driver of FSC acquisition and fibrogenesis. Upregulation and colocalization of SOX2 and COL1 were found in the fibrotic lung tissues of CNT-exposed mice via oropharyngeal aspiration after 56 days. The knockdown of SOX2 by gene silencing abrogated the fibrogenic and FSC-inducing effects of CNTs. Chromatin immunoprecipitation assays identified SOX2-binding sites on COL1A1 and COL1A2, indicating SOX2 as a transcription factor in collagen synthesis. SOX2 was also found to play a critical role in TGF-ß-induced fibrogenesis through its collagen- and FSC-inducing effects. Since many nanomaterials are known to induce TGF-ß, our findings that SOX2 regulate FSCs and fibrogenesis may have broad implications on the fibrogenic mechanisms and treatment strategies of various nanomaterial-induced fibrotic disorders.

Predicting Nanotube Fibrogenicity through Stem Cell-Mediated Fibroblast Focus and Spheroid Formation.

Fibroblast stem cells or stemlike cells (FSCs) are proposed to play a pivotal role in extracellular matrix (ECM) regeneration by serving as a key source of ECM-producing fibroblasts. We developed a mechanism-based in vitro model for fibrogenicity testing of nanomaterials based on their ability to induce FSCs. Using a FSC-enriched fibroblast focus model to mimic in vivo fibrogenic response, we demonstrated a dose-dependent increase in fibroblast focus formation and collagen production by primary lung fibroblasts treated with multiwalled carbon nanotubes (MWCNTs). The focus-forming cells exhibited stem properties as indicated by stem cell markers expression, sphere formation, and ALDH activity assays. Inhibition of ALDH activity diminished the focus and sphere formation as well as collagen production. In vivo animal studies supported the in vitro findings and indicated the potential utility of FSC-based assays as a rapid screening tool for fibrogenicity testing of nanomaterials. This study also unveils a novel mechanism of nanotube-induced fibrogenesis through ALDH-dependent FSC activation.

A paradoxical response of the rat organism to long-term inhalation of silica-containing submicron (predominantly nanoscale) particles of a collected industrial aerosol at realistic exposure levels.

While engineered SiO2 nanoparticle toxicity is being widely investigated, mostly on cell lines or in acute animal experiments, the practical importance of as well as the theoretical interest in industrial condensation aerosols with a high SiO2 particle content seems to be neglected. That is why, to the best of our knowledge, long-term inhalation exposure to nano-SiO2 has not been undertaken in experimental nanotoxicology studies. To correct this data gap, female white rats were exposed for 3 or 6 months 5 times a week, 4h a day to an aerosol containing predominantly submicron (nanoscale included) particles of amorphous silica at an exposure concentration of 2.6±0.6 or 10.6±2.1mg/m3. This material had been collected from the flue-gas ducts of electric ore smelting furnaces that were producing elemental silicon, subsequently sieved through a<2µm screen and redispersed to feed a computerized "nose only" inhalation system. In an auxiliary experiment using a single-shot intratracheal instillation of these particles, it was shown that they induced a pulmonary cell response comparable with that of a highly cytotoxic and fibrogenic quartz powder, namely DQ12. However, in long-term inhalation tests, the aerosol studied proved to be of very low systemic toxicity and negligible pulmonary fibrogenicity. This paradox may be explained by a low SiO2 retention in the lungs and other organs due to the relatively high solubility of these nanoparticles. nasal penetration of nanoparticles into the brain as well as their genotoxic action were found in the same experiment, results that make one give a cautious overall assessment of this aerosol as an occupational or environmental hazard.