Amruta Manke

Date of Graduation

2015

Document Type

Dissertation

Degree Type

PhD

College

School of Pharmacy

Department

Pharmaceutical Sciences

Committee Chair

Yon Rojanasakul

Committee Co-Chair

Patrick Callery

Committee Member

Vincent Castranova

Committee Member

Grazyna Szklarz

Committee Member

Liying Wang

Abstract

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.

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