Stiffness: a master regulator of fibrogenesis?
AuthorMitke, Asres Berhan
AffiliationPharmacology and Therapeutics
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2021-04-29.
© 2019 Dr. Asres Berhan Mitke
Fibrosis is one of the leading causes of death that may affect all organs in the human body. All forms of fibrosis are characterized by fibrotic scarring resulting from replacement of normal tissue with extracellular matrix (ECM). Idiopathic pulmonary fibrosis (IPF) is the most common form of idiopathic interstitial pneumonias; it is relentlessly progressive and ultimately fatal. The aberrant accumulation of myofibroblasts and the excessive deposition of ECM are the main features of IPF. The suggested sources of myofibroblasts include resident lung fibroblasts, lipofibroblasts, epithelial-mesenchymal transition (EMT), endothelial-mesenchymal transition (EndoMT) and recruitment from circulating fibrocytes. Lipofibroblasts are lipid-droplet containing cells, which have a key role in maintaining homeostasis of the lung. However, IPF patients have stiffer lungs and homeostatically dysregulated cellular microenvironments. Stiff substrates are known to augment myofibroblast differentiation, ECM production and the activation of the prototype profibrotic cytokine, transforming growth factor β1 (TGF-β1). In contrast, stiff substrates inhibit cyclooxygenase-2 (COX-2) expression and the synthesis of the anti-fibrotic prostanoid, prostaglandin E2 (PGE2). This thesis for the first time revealed a reduction in the lipofibroblast population and prostaglandin E synthase (PTGES) level in the lungs of IPF patients. This current study also highlighted the significance of dimensionality in modulating fibroblast behavior by establishing human lung fibroblasts in 2D soft and 3D soft microenvironments (that have similar stiffness) and performing head-to-head comparisons with the conventional 2D stiff cultures. Our data demonstrated the marked suppression of fibroblast proliferation and fibrogenesis in 3D soft microenvironment, as contrasted with 2D soft, independent of differences in prostanoid levels. This thesis also presented evidence that suggested the augmented anti-fibrogenic actions of glucocorticoid and PGE2 combination. Within this thesis, I showed that the phenotypic plasticity of fibroblasts depends on substrate stiffness and dimensionality. The level of lipid-droplet inclusions that mark the formation of lipofibroblasts was remarkably increased in 3D fibroblasts. In 2D soft and 3D microenvironments, striking reductions of the myofibroblast marker, alpha smooth muscle actin (ACTA2) and increases in the lipofibroblast markers, adipose differentiation-related protein (ADRP) were observed. Activated exogenous TGF-β1, despite elevating expression of the fibrogen interleukin-11 (IL-11), did not induce the differentiation of lipofibroblast into myofibroblasts in 2D soft 3D soft settings. This study has not only provided insight into fibroblast phenotypic plasticity, but has also revealed a novel role of parathyroid hormone-related protein (PTHrP), as a main effector in mediating the effects of softness or dimensionality on fibroblast phenotypic plasticity. Moreover, this study has established the anti-fibrogenic actions of PTHrP in lung fibroblasts in vitro.
Keywordsfibrosis; lung; 3D, 2D soft; idiopathic pulmonary fibrosis; stiffness
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