Master of Science
Physiology and Pharmacology
Gill, Sean E.
Pulmonary microvascular endothelial cell (PMVEC) interactions with the extracellular matrix (ECM) mediates PMVEC barrier function. Further, data suggests an association between decreased PMVEC-ECM interactions under proinflammatory conditions and PMVEC barrier dysfunction. Tissue inhibitor of metalloproteinases 3 (TIMP3) may also regulate barrier function, as PMVEC from Timp3-/-mice show increased leak. Studies in the developing lung also showed TIMP3 regulating PMVEC-ECM interactions. Based on this, I hypothesized that TIMP3 maintains PMVEC barrier function by promoting PMVEC-ECM interactions.
Using the XperT-permeability assay, Timp3-/-PMVEC demonstrated enhanced leak vs. WT PMVEC, particularly under proinflammatory conditions, and this was associated with decreased phosphorylated focal adhesion kinase (pFAK). Furthermore, FAK inhibition increased leak in WT PMVEC, and enhanced leak in Timp3-/-PMVEC under proinflammatory conditions. Finally, MMP inhibition attenuated leak in Timp3-/-PMVEC under both basal and proinflammatory conditions
These results suggest a role for TIMP3 in mediating PMVEC-ECM interactions and promoting PMVEC barrier function.
Summary for Lay Audience
Endothelial cells (EC) line the blood vessels and are important in maintaining their function. EC normally form a barrier by interacting with each other and with the supporting scaffold found beneath the EC, known as extracellular matrix (ECM). A specific protein called tissue inhibitors of metalloproteinases 3 (TIMP3) may help support EC in their role of maintaining a barrier within the lungs as an absence of TIMP3 is shown to increase fluid buildup in the lungs due to EC losing their ability to form a barrier. While exactly how TIMP3 regulates EC in the lung is still unclear, it is possible that TIMP3 regulates the interaction of EC with their environment. As such, I hypothesized that TIMP3 regulates lung function by supporting the interaction of EC with the ECM.
By comparing normal mouse lung cells with cells that contained no TIMP3 (Timp3-/- cells), I found that the Timp3-/- cells had fewer focal adhesion proteins in them, as compared to the normal cells. Focal adhesions are important proteins found inside cells that help maintain the interaction of cells with their environment. Additionally, the Timp3-/- cells seemed to be leakier than the normal cells, as shown by a fluorescent protein that marked where gaps would form between the cells and their environment. Addition of inhibitors that targeted metalloproteinases, proteins that TIMP3 is shown to inhibit within the tissue, rescued the increased leak in normal cells as well as in the Timp3-/- cells. Furthermore, after adding inhibitors to block focal adhesion formation, both normal cells and Timp3-/- cells showed an increase in leak. These results were especially prominent in groups where an inflammatory environment was induced.
These results show a promising start to understanding the role of TIMP3 in mediating cellular interaction with each other and with the surrounding environment in the lungs. Importantly, they suggest that TIMP3 and EC-ECM interactions may be a possible therapeutic target for supporting the EC barrier function under inflammatory conditions in the lungs.
Kulkarni, Nidhi, "The Role of TIMP3 in Microvascular Endothelial Cell-Extracellular Matrix Interaction and Regulation of Microvascular Barrier Function" (2020). Electronic Thesis and Dissertation Repository. 7490.
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Available for download on Friday, December 09, 2022