| Endothelial cells comprise the nearly impermeable single cell barrier that lines the lumen of all blood vessels. During angiogenesis, endothelial cells migrate from an existing capillary into the surrounding extracellular matrix to ultimately form a new capillary. Physiologically, blood vessel formation is important for wound healing; however, aberrant endothelial cell behavior can lead to tumor formation and numerous vascular diseases. This thesis extends the current knowledge of endothelial cell adhesion and migration by investigating the traction forces endothelial cells exert on their substrate. We are the first to apply Traction Force Microscopy (TFM), a technique that quantifies the magnitude, direction and spatial location of the traction forces exerted by a cell on its substrate, to the study of endothelial cell adhesion. We have created well-characterized substrates to investigate changes in endothelial cell traction resulting from changes in ligand density and mechanical compliance. Using TFM, we have determined the relationship between cell force and area over a wide range of ligand concentrations, and have found fundamental differences in cell behavior. Our results indicate the total magnitude of force that a cell exerts is related to its area. Moreover, we characterized the effects of ligand density on the ability of endothelial cells to adhere and exert forces immediately after plating and throughout spreading. Ligand density, in part, dictates the rate of spreading, the extent of spreading and the shape changes that the cells undergo during spreading. To further probe the relationship between traction and cell area, we also investigated the cellular adhesion and contractile mechanisms of focal adhesion and stress fiber formation during spreading. Interestingly, we found that focal adhesion, as marked by vinculin clustering, and actin stress fibers, as marked by phalloidin staining, are not necessary for traction force generation. In a first step towards understanding tissue formation, we explored endothelial cell-cell cohesion by investigating the changes cellular traction during cell-cell contact. Interestingly, we found that cell contact results in both global and local changes in traction generation. Even more notably, we identified a previously undescribed form of cell-cell communication where cells sense and respond to adjacent cells through traction-driven tension created in the substrate. This data coincides with recent studies of durotaxis and demonstrate a novel mechanism of cell-cell communication. These insights into the mechanics of endothelial cell behavior will ultimately benefit the rational design of biomaterials and tissue-engineered therapeutics. |
