Role of Stem-like Cells in Carbon Nanotube-Induced Pulmonary Fibrosis

Recent studies have shown that pulmonary exposure to (CNT) results in rapid and progressive interstitial lung fibrosis in animals without causing persistent lung inflammation, which is normally associated with other known fibrogenic agents. This unusual fibrogenic effect of CNT raises important health issues since the exposure could result in deadly and incurable lung fibrosis. Accumulating evidence indicates the fibrogenic potential of carbon nanotubes, however, the underlying mechanism remains poorly addressed. Recent studies have demonstrated the pathogenic role of mesenchymal stem cells in pulmonary fibrosis that differentiate into myofibroblasts and contribute to disease progression. Understanding the molecular/cellular basis of these fibrosis-associated stem cells during lung fibrosis is of critical importance. However, the concept of stemness in the light of nanomaterial-induced fibrosis remains to be explored. Fibroblast cells being the key players in fibrogenesis, we hypothesized that CNT exposure in fibroblasts induce fibroblast stem-like cells (FSCs) which are critical for the CNT-induced fibrogenic response. The long-term broad objective of this project was to develop an in vitro model predictive of in vivo fibrogenic response and to devise preventive strategies for the disease. The specific aims of this study included i) Determining the involvement of stemness phenotype and underlying mechanism in CNT-induced lung fibrosis the and develop in vitro screening assay which may be predictive of the in vivo fibrogenic response; ii) Investigate the redox regulation of stem-like cells involved in CNT-driven fibrosis; iii) Evaluating the impact of nanoparticle length and surface chemical modification influence stemness phenotype and the resulting fibrogenic response. Our findings from Aim 1 indicated that indeed CNTs induced the side population phenotype (indicative of the fibroblast stem-like cell phenotype) of primary lung fibroblasts. The isolated FSCs displayed an elevated expression of fibrogenic and stem cell markers indicating the reliability of the stem cell isolation method as well as supporting their role in CNT-induced fibrogenesis. The study also developed and put forth an in vitro model of CNT-induced fibrotic nodule formation that correlates the development of stemness phenotype and onset of fibrosis. Furthermore, the results from Aim 2 demonstrated that CNT-induced stemness phenotype was under the redox regulation via identifying the key role of peroxides in CNT-induced FSC generation and collagen expression. Moreover, results from our second study revealed that antioxidants abrogated the effect of CNT on stem-like cell generation suggesting crucial role of redox in stemness generation and the fibrogenic effects. Our outcomes from the Aim 3 demonstrated a length-dependent effect on stemness phenotype, with longer CNT inducing higher FSCs compared to short CNTs as evidenced by side population and aldehyde dehydrogenase assays. Pristine CNTs induced higher FSCs compared to modified CNTs; however the effect was not statistically different. Long SWCNTs induced greater fibrogenic response in vivo compared to short SWCNTs, supporting the potential utility of our in vitro FSC model to predict the fibrogenicity of CNTs. Such information will be important for development and safer design and use of nanotechnology.;Findings from this work introduced the concept of fibroblast stem-like cells as a potential key player in the pathogenesis of pulmonary fibrosis; which in turn may help in identifying novel biomarkers and drug targets for early diagnosis and treatment of the disease. Furthermore, the in vitro FSC model developed in this study may be utilized as a rapid screening tool for fibrogenicity testing of not just carbon nanomaterials but also other nanoparticles and antifibrotic agents

Effect of Fiber Length on Carbon Nanotube-Induced Fibrogenesis

Given their extremely small size and light weight, carbon nanotubes (CNTs) can be readily inhaled by human lungs resulting in increased rates of pulmonary disorders, particularly fibrosis. Although the fibrogenic potential of CNTs is well established, there is a lack of consensus regarding the contribution of physicochemical attributes of CNTs on the underlying fibrotic outcome. We designed an experimentally validated in vitro fibroblast culture model aimed at investigating the effect of fiber length on single-walled CNT (SWCNT)-induced pulmonary fibrosis. The fibrogenic response to short and long SWCNTs was assessed via oxidative stress generation, collagen expression and transforming growth factor-beta (TGF-β) production as potential fibrosis biomarkers. Long SWCNTs were significantly more potent than short SWCNTs in terms of reactive oxygen species (ROS) response, collagen production and TGF-β release. Furthermore, our finding on the length-dependent in vitro fibrogenic response was validated by the in vivolung fibrosis outcome, thus supporting the predictive value of the in vitro model. Our results also demonstrated the key role of ROS in SWCNT-induced collagen expression and TGF-β activation, indicating the potential mechanisms of length-dependent SWCNT-induced fibrosis. Together, our study provides new evidence for the role of fiber length in SWCNT-induced lung fibrosis and offers a rapid cell-based assay for fibrogenicity testing of nanomaterials with the ability to predict pulmonary fibrogenic response in viv

Effect of Fiber Length on Carbon Nanotube-Induced Fibrogenesis

Given their extremely small size and light weight, carbon nanotubes (CNTs) can be readily inhaled by human lungs resulting in increased rates of pulmonary disorders, particularly fibrosis. Although the fibrogenic potential of CNTs is well established, there is a lack of consensus regarding the contribution of physicochemical attributes of CNTs on the underlying fibrotic outcome. We designed an experimentally validated in vitro fibroblast culture model aimed at investigating the effect of fiber length on single-walled CNT (SWCNT)-induced pulmonary fibrosis. The fibrogenic response to short and long SWCNTs was assessed via oxidative stress generation, collagen expression and transforming growth factor-beta (TGF-β) production as potential fibrosis biomarkers. Long SWCNTs were significantly more potent than short SWCNTs in terms of reactive oxygen species (ROS) response, collagen production and TGF-β release. Furthermore, our finding on the length-dependent in vitro fibrogenic response was validated by the in vivo lung fibrosis outcome, thus supporting the predictive value of the in vitro model. Our results also demonstrated the key role of ROS in SWCNT-induced collagen expression and TGF-β activation, indicating the potential mechanisms of length-dependent SWCNT-induced fibrosis. Together, our study provides new evidence for the role of fiber length in SWCNT-induced lung fibrosis and offers a rapid cell-based assay for fibrogenicity testing of nanomaterials with the ability to predict pulmonary fibrogenic response in vivo

Potential in vitro model for testing the effect of exposure to nanoparticles on the lung alveolar epithelial barrier

Pulmonary barrier function plays a pivotal role in protection from inhaled particles. However, some nano-scaled particles, such as carbon nanotubes (CNT), have demonstrated the ability to penetrate this barrier in animal models, resulting in an unusual, rapid interstitial fibrosis. To delineate the underlying mechanism and specific bio-effect of inhaled nanoparticles in respiratory toxicity, models of lung epithelial barriers are required that allow accurate representation of in vivo systems; however, there is currently a lack of consistent methods to do so. Thus, this work demonstrates a well-characterized in vitro model of pulmonary barrier function using Calu-3 cells, and provides the experimental conditions required for achieving tight junction complexes in cell culture, with trans-epithelial electrical resistance measurement used as a biosensor for proper barrier formation and integrity. The effects of cell number and serum constituents have been examined and we found that changes in each of these parameters can greatly affect barrier formation. Our data demonstrate that use of 5.0 × 104 Calu-3 cells/well in the Transwell cell culture system, with 10% serum concentrations in culture media is optimal for assessing epithelial barrier function. In addition, we have utilized CNT exposure to analyze the dose-, time-, and nanoparticle property-dependent alterations of epithelial barrier permeability as a means to validate this model. Such high throughput in vitro cell models of the epithelium could be used to predict the interaction of other nanoparticles with lung epithelial barriers to mimic respiratory behavior in vivo, thus providing essential tools and bio-sensing techniques that can be uniformly employed

Induction of Stemlike Cells with Fibrogenic Properties by Carbon Nanotubes and Its Role in Fibrogenesis

We developed a three-dimensional fibroblastic nodule model for fibrogenicity testing of nanomaterials and investigated the role of fibroblast stemlike cells (FSCs) in the fibrogenic process. We showed that carbon nanotubes (CNTs) induced fibroblastic nodule formation in primary human lung fibroblast cultures resembling the fibroblastic foci in clinical fibrosis and promoted FSCs that are highly fibrogenic and a potential driving force of fibrogenesis. This study provides a predictive 3D model and mechanistic insight on CNT fibrogenesis

Blocking Alcoholic Steatosis in Mice with a Peripherally Restricted Purine Antagonist of the Type 1 Cannabinoid Receptor

Type 1 cannabinoid receptor (CB1) antagonists have demonstrated promise for the treatment of obesity, liver disease, metabolic syndrome, and dyslipidemias. However, the inhibition of CB1 receptors in the central nervous system can produce adverse effects, including depression, anxiety, and suicidal ideation. Efforts are now underway to produce peripherally restricted CB1 antagonists to circumvent CNS-associated undesirable effects. In this study, a series of analogues were explored in which the 4-aminopiperidine group of compound <b>2</b> was replaced with aryl- and heteroaryl-substituted piperazine groups both with and without a spacer. This resulted in mildly basic, potent antagonists of human CB1 (hCB1). The 2-chlorobenzyl piperazine, <b>25</b>, was found to be potent (<i>K</i>_i = 8 nM); to be >1000-fold selective for hCB1 over hCB2; to have no hERG liability; and to possess favorable ADME properties including high oral absorption and negligible CNS penetration. Compound <b>25</b> was tested in a mouse model of alcohol-induced liver steatosis and found to be efficacious. Taken together, <b>25</b> represents an exciting lead compound for further clinical development or refinement