| Idiopathic Pulmonary Fibrosis (IPF) afflicted lungs are burdened with an over-population of activated fibroblasts, which are believed to be the central orchestrators of this disease. These cells are instrumental in deposition of excessive extra-cellular matrix, leading to destruction of lung architecture and terminal dysfunction. This dissertation details a multi-prong approach undertaken to investigate the biology of primary fibroblasts derived from the lungs of patients with (IPF-F) in comparison to their normal counterparts (normal-F) that were either frozen immediately post-isolation (non-cultured) or continued in vitro (cultured). The proliferative profile of IPF-F is investigated in detail, and the sensitivity of IPF-F to a series of bioactive compounds namely heparin, thalidomide and curcumin is evaluated in vitro. In addition, the dysregulation of the microfilament cytoskeleton in IPF-F is investigated via the gene and protein expression analysis of beta-actin and the actin-monomer sequestering protein, thymosin-beta4 (Tbeta4). No net significant difference was observed in the rate of proliferation or expression of proliferation marker between in vitro cultured IPF-F and normal-F. However, significantly higher levels of proliferation marker were observed in the non-cultured IPF-F compared to non-cultured normal-F. Since non-cultured potentially represent a phenotype closer to in vivo state than the in vitro cultured cells, we conclude that IPF-F potentially possess a higher rate of proliferation in vivo, and that a higher rate of proliferation may account for the fibroblast over-abundance. Imunohistochemistry analysis revealed higher levels of proliferation marker in parenchymal areas surrounding the fibrotic areas and fibroblast foci, but unexpectedly lower levels within the fibroblast foci and the advanced areas of fibrosis. These fibrotic areas and fibroblast foci also expressed high of the fibroblast activation marker, alpha-SMA. We propose that cell proliferation occurs in the quasi-normal non-fibrotic regions of the lung, and an alternative mechanism, such as increased migration to the foci, may lead to fibroblast accumulation in the foci. The in vitro drug-sensitivity analysis revealed mixed results for heparin with no net efficacy, while thalidomide exhibited no sensitivity. Curcumin depicted a high sensitivity and specificity for fibroblasts with comparative insensitivity of the epithelial cells, and merits further detailed analyses. The analysis of the micro-filament proteins revealed significantly higher levels of beta-actin protein in non-cultured IPF-F compared to non-cultured normal-F, however, mRNA levels of beta-actin were lower in IPF-F. We propose that significantly higher levels of Tbeta4 may play a role in beta-actin protein accumulation. In summary, this dissertation provides extensive information on the biological behavior of IPF-F and provides a resource for the role of fibroblasts in the diseased IPF lung. |
