Study of Reactive Dicarbonyl Species-Induced Epithelial to Mesenchymal Transition in Diabetes Exacerbating Pulmonary Fibrosis (Ii)－Clarification of Age/Rage-Independent Pathways and Analysis of Correlation with Clinical Outcome

Diabetes mellitus was known to be an independent risk factor for the exacerbation of pulmonary fibrosis. However, instead of the critical role of advanced glycation end products (AGE)/receptor of AGE (RAGE)-pathway, a major pathogenic pathway in hyperglycemia, in the fibrosis of nephropathy and cardiomyopathy, it was found to have anti-fibrotic and anti-epithelial to mesenchymal transition (EMT) properties in pulmonary fibrosis. These studies indicate an AGE/RAGE-independent pathway in diabetes exacerbating pulmonary fibrosis. Reactive dicarbonyls including methylglyoxal (MG), glyoxal and 3-deoxyglucosone (3-DG) were known to be involved in the pathology of diabetic complications by direct modification of proteins, nucleotides or phospholipids. In the first year, we have found that the treatment of high glucose, MG or glyoxal but not AGE significantly induced EMT in human alveolar epithelial cells (HAECs). The treatment of AGE even revealed anti-EMT activity. Moreover, MG-increased vimentin was significantly reversed by the transfection of glyoxalase 1, a major metabolic enzyme of MG and glyoxal. We have also found the critical role of reactive dicarbonyls caused protein misfolding and subsequent ER stress in reactive dicarbonyls-induced EMT. On the other hand, according to the data of RNA seq, MIR566, MIR564, MIR221, MIR614, MIR34AHG, MIR205HG and MIR25 may be potential targets in MG-exacerbating pulmonary fibrosis. In addition, we have established a mice model of bleomycin-induced pulmonary fibrosis, and found exacerbating pulmonary fibrosis in streptozotocin-diabetic mice. We have also collected and analyzed the pleural effusions of patients, 78.5% diabetic patients revealed higher expression of MG-modified proteins in pleural effusion, while that of non-diabetic patients was only 25%. This data implied a possible role of active dicarbonyl and its modified proteins in lung diseases. In the following project, more experiments will be performed to clarify the AGE/RAGE-independent mechanisms of reactive carbonyls-induced EMT in diabetes exacerbating pulmonary fibrosis. The correlation between the levels of reactive dicarbonyls in plasma and sputum and lung function in pulmonary fibrosis patients will be examined. In addition, the scavenging activity of reactive dicarbonyls of clinical drugs and natural products will be examined for develop potential candidates for treating diabetes exacerbating pulmonary fibrosis and other complications of diabetes. Specific aim 1: To clarify the mechanisms of reactive dicarbonyls-induced EMT in diabetes exacerbating pulmonary fibrosis. Aim 1: To define the role of reactive dicarbonyls-induced EMT as an AGE/RAGE independent pathway in diabetes exacerbating pulmonary fibrosis. Aim 2: To determine the mechanism of ER stress regulated reactive dicarbonyls-induced EMT, and discover a comprehensive proteomic analysis of reactive dicarbonyls-modified proteins by using iTRAQ labeling method and characterize their function in EMT by STRING software. Aim 3: To confirm the identified miRNAs that indeed participate in the regulation of reactive dicarbonyls-driven EMT. Aim 4: To examine the effects of these potential therapeutic targets in the animal model of bleomycin-induced lung fibrosis. Specific aim 2: To define reactive dicarbonyl as a predictor or potential therapeutic objective for the exacerbation of pulmonary fibrosis in diabetic patients. Aim 1: To analyze the correlation between the changes of reactive dicarbonyls levels in plasma and sputum and the exacerbation of pulmonary fibrosis in patients. Aim 2: To compare the expression of reactive dicarbonyls-induced EMT in normal and diseased human airway epithelial cells, and investigate the possible mechanism by DNA microarray. Aim 3: To screen the reactive dicabonyls scavenging activity of FDA approved drugs and natural products for treating diabetes exacerbating pulmonary fibrosis and other diabetic complications.