Role of Phospholipase D in Idiopathic Pulmonary Fibrosis and FTIR Imaging to Detect Fibrogenesis
thesisposted on 2018-11-27, 00:00 authored by Vidyani Suryadevara
Idiopathic pulmonary fibrosis (IPF) is a pernicious lung disease characterized by scar formation and respiratory failure. This is due to dysregulated repair of the epithelial injury in the lung, which involves epithelial apoptosis, followed by activation of fibroblasts to mediate myofibroblasts, thereby leading to the deposition of the extracellular matrix in the lung. Currently there are only two FDA approved drugs for IPF; which do not cure the disease, but just slow the progression of disease, there is a need to identify new therapeutic targets for the disease. Phospholipase D (PLD), an important phospholipid metabolizing enzyme me involved in several pathophysiologies, catalyzes the hydrolysis of phosphatidylcholine (PC), generating phosphatidic acid (PA) and choline. In addition to PC, PLD also hydrolyzes other phospholipids such as phosphatidylethanolamine (PE) and cardiolipin (CL). PLD mediated PA generation is involved in regulation of various cellular processes including cell survival, cell migration, cell proliferation, differentiation, cytoskeletal changes, membrane trafficking, and autophagy. Previous studies have shown that PLD has a role in cardiac and hepatic fibrosis, but the mechanism(s) by which PLD mediates fibrosis has not been identified. This study is aimed at delineating the PLD signaling mechanism in IPF to identify new therapeutic targets. Of the two PLD isoenzymes, PLD1 and PLD2, the protein expression of PLD2 was up-regulated in bleomycin induced pulmonary fibrosis (PF), and PLD2 deficient mice (Pld2-/- null) were protected against bleomycin induced lung inflammation and fibrosis, thereby establishing the role of PLD in IPF. To further understand how PLD mediates epithelial injury during IPF, bronchial airway epithelial cell line (Beas2B cells) was used, and in vitro studies showed that bleomycin challenge led to enhanced PLD activity in the cells; led to generation of mitochondrial reactive oxygen species (ROS) and apoptosis in Beas2B cell line. Further, inhibition of PLD2 in Beas2B cells attenuated bleomycin induced mitochondrial DNA damage and epithelial cell apoptosis. Thus, we identified that reducing bleomycin-induced PLD activity will lessen mitochondrial ROS generation, thereby leading to less mitochondrial dysfunction and eventually less apoptosis of the epithelial cells. This study identifies PLD to be a promising therapeutic target in IPF. In addition to identifying new therapeutic targets, the current research study is focused on developing new diagnostic techniques for IPF. Fourier Transform Infrared (FT-IR) imaging technique is a label-free, non-destructive approach to detect histopathological changes in the tissue. IR spectra has been acquired from the lungs from bleomycin challenged mice harvested at different time-points. The biochemical changes that occur in the lung tissue during the development of fibrosis can be detected by this technique, based on the spectral profile acquired. The spectral data obtained is subjected to unsupervised cluster analysis which classifies it into based on the histopathological states. The ratio of absorption of spectral ratios gives the collagen map, glycosylation and structural deformations across the lung tissue. Linear discriminant analysis was done to classify and identify the extent of disease progression. This novel technique gives the biochemical information across the entire lung tissue and enables diagnosis of the disease in a label-free manner.