Over the summer, I had the opportunity to work on a project that investigates the microbiology of the disease progression of pulmonary fibrosis. Pulmonary fibrosis is a chronic lung disease that is caused by the hardening of the lung tissue, preventing the lung from functioning properly. This progressively makes it harder for the patient to breathe properly. It is a disease that affects 5 million people worldwide, with a median survival rate of 2-3 years. There is currently no known cure for the disease. The most common type of pulmonary fibrosis is known as idiopathic pulmonary fibrosis. This means that the onset of the disease is unknown, making it even more difficult to find treatment for the patient that is affected. The most that can be done for a patient with pulmonary fibrosis are therapies and medications that alleviate the symptoms that the disease brings. Pulmonary fibrosis changes the lung’s anatomy when there is an overproduction of the extracellular matrix. This happens when proteins such as collagen are produced in excess by myofibroblasts. This extra collagen causes the hardening and thickening of the lung tissues that prevent the alveoli from expanding and contracting appropriately, and prevents oxygen exchange from occurring properly as well.
The project that I had the opportunity to work with looked into the role that endothelial cells play in the progression of pulmonary fibrosis, specifically focusing on the role of endothelial cell derived Thrombospondin-1. Endothelial cells make up the endothelium, which is the inner lining of blood vessels. We look into TSP1 because it is known as a multifunctional cellular angiogenesis inhibitor and is known to interact with cytokines such as TGF-beta.
Prior to my work in the lab, the Post-Doc that I was working with found that there was no correlation between TSP-1 knockout mice, and an expression of fibroblasts, which are the widely accepted molecules, in current literature, that contribute to the excess collagen that is characteristic of pulmonary fibrosis. However, it was discovered that there was an increase in expression of metallo-matrixproteinases (MMPs) by TSP1 knockout lung endothelial cells. This is significant because metallo-matrixproteinases are known to be important drivers of a process called epithelial-mesenchymal transition, in which epithelial cells of the body obtain mesenchymal, or fibroblast-like properties. This lead to my hypothesis that the increase in pulmonary fibrosis found in TSP1 knockout lung endothelial cells may be due to an increase in EMT. The general hypothesis was that TSP1 binds to and deactivates MMPs and therefore prevents a large driver of EMT from activating. More specifically, I hypothesized that EMT will occur at a greater rate in the epithelial cells plated in TSP1-deficient endothelial cell conditioned media than when plated in wild-type endothelial cell conditioned media.
To test this, I performed an invasion assay, which is a procedure in which the cell to be tested is plated on top of a membrane in serum-free media, and then attracted to come through the pores of the membrane using a chemoattractant. I used to this to see the difference in cell motility, which is another characteristic of EMT. In my experiment, I plated wild-type epithelial cells on top of the membrane in serum-free media conditioned with either wildtype or TSP1 knockout endothelial cells, and then used 15% serum media as the chemoattractant. We then stained with Crystal Violet to locate cells that had migrated through the membrane. We found that more epithelial cells migrated through the membrane when they were plated in TSP1 knockout serum free conditioned media than when plated in the wildtype serum free conditioned media. We also saw a difference in morphology in all cells tested. On the top of the membrane, they formed epithelial cell colonies, whereas on the bottom of the membrane, they formed mesenchymal morphologies. This suggests that EMT is occurring, and at a faster rate in TSP1 knockout. We can use the results of this project to study the role of endothelial cell derived TSP1 in cancer metastasis.