It is estimated that more than 1 bn people suffer from acute and chronic lung problems resulting in millions of deaths every year. The respiratory disorders with the most significant impact on public health are asthma, COPD, lung cancer and lung infections1.
Cells must communicate with each other for the human body to function. Cells usually exchange messages by secreting proteins and other molecules, which bind to other cells and influence their behaviour. However, in recent years it is becoming clear that the cells are also able to package their products into small membrane-enclosed vesicles and send them to target cells elsewhere. These vesicles play a role in pathologies such as cancer, cardiovascular disease and neurodegenerative disease, but their functions in lung and lung disease are not well understood. This project focuses on the question how small extracellular vesicles (EVs) are released from lung cells, how they influence the neighbouring cells and how their role changes in pulmonary disease such as lung cancer. This study will contribute to the knowledge necessary for development of new therapies for respiratory disorders.
EVs are commonly divided in microvesicles and exosomes, which have different mechanisms of generation and release. Microvesicles are shed directly from the plasma membrane, whereas exosomes are generated in intracellular endosome-derived vesicles called multivesicular bodies2. Initially it was thought that formation and release of EVs is merely a cellular waste-disposal mechanism. However, recent research suggests that the cell loads selected contents in nascent EVs and that the concentration of nucleic acids and proteins inside the vesicles can differ considerably from their concentration in the cytosol2. EVs from different cell types are heterogenous and even those from the same cell type can differ depending on the cell functional state. Some EVs rupture soon after secretion and their contents act locally. But the membrane of most vesicles stays intact, and the vesicles are passively transported to other cells via extracellular fluid2. In some cases, binding of EVs to receptors on the cell membrane is enough to trigger the activation of second messenger cascades inside the cell. However, EVs are mostly taken up by endocytosis or they fuse directly with the recipient cell plasma membrane3. The major effector mechanism of EVs is thought to be luminal miRNA, which influences gene expression and function of recipient cells. The molecular mechanisms of EV release and uptake in the recipient cell are not clear.
Similarly, there is little information available about the role of EVs in the lungs. Lung EVs were initially suggested to play a role in the immune response and in antigen presentation due to their MHC protein content. This hypothesis was further strengthened by the evidence that EVs isolated from asthmatic individuals could promote immune response. In addition to the role of EVs in asthma, it has also been shown that exosome secretion is increased in COPD. EVs secreted from lung macrophages have been investigated in further detail and it was shown that they promote inflammation in the lungs in response to cigarette smoke and other types of lung injury4. Release of EVs from lung cancer cells is also an area of active investigation. However, although there is clear evidence of cell-cell communication via EVs in lung disease, the function and mechanisms of extracellular vesicle secretion from healthy lung epithelial cells is not clear.
The aim of this study is to investigate EV secretion and uptake in lung cells and to better understand EV function in normal lung epithelial cells and lung cancer cells.
The objectives to achieve this aim are:
Objective 1: Characterisation of EVs secreted from lung epithelial cells
Previous studies suggested that EVs in broncho-alveolar lavage fluid are mostly released from lung epithelial cells. The EVs from epithelial cells will be isolated and quantified by measuring total particle number and total protein5. Isolated EVs will be characterised by detection of positive EV markers such as tetraspanins and EV cytosolic proteins.
Objective 2: Mechanisms of EV secretion from lung cells.
RNA-specific nucleic dyes that fluorescently label intracellular compartments with high RNA content, but only weakly bind to DNA will be used to investigate multivesicular bodies in lung epithelial and cancer cells. In addition, fluorescent dyes with structure similar to membrane sphingolipids, such as Bodipy TR ceramide, will be used for EV labelling. Previously published work suggests that EV secretion is mediated by intracellular increase in Ca2+ concentration and by protein kinase C (PKC) activation. Both pathways can be triggered with extracellular ATP. To further establish the most relevant signalling pathway for multivesicular body exocytosis, Ca2+ ionophore and PKC activator will be used. In addition to fluorescence microscopy, secretion of fluorescently labelled EVs from lung epithelial cells and lung cancer cells will be investigated with biochemical methods. Together, these experiments will deliver information on the signalling pathways leading to EV secretion in the lung.
Objective 3: Fibroblast EV uptake and function in lung epithelial cells
It was reported previously that EVs from mesenchymal stem cells promote lung cell proliferation and migration in wound healing4. It is also known that interaction between lung epithelial cells and fibroblasts regulates epithelial to mesenchymal transition and influences lung remodelling. However, the uptake and function of fibroblast EVs in lung epithelial cells is not well known. EVs will be isolated from cultured fibroblasts using commercially available isolation and purification kits. Isolated EVs will be transferred to cultured epithelial cells and EV uptake will be quantified using fluorescence microscopy. The effect of EVs on lung cell proliferation and migration will be investigated using methods such as MTT assay, viable cell count and scratch essay.