To generate replacement blood vessels, it is useful to understand how cellular processes are integrated to assemble blood vessels in the body. However, our knowledge of these integrating links is sparse. Blood vessel formation occurs via the actions of endothelial cells that include both cell division and morphogenetic processes such as adhesion and migration. Normally these distinct processes are elegantly interwoven to produce the appropriate amount of vasculature with the proper 3-dimensional pattern. Most angiogenic agents activate signaling pathways that affect both division and morphogenesis, but how this occurs is not entirely clear. This work will investigate these relationships, and test the central hypothesis that control of endothelial cell division is linked to crucial morphogenetic events that result in new blood vessels. This will be done using dynamic image analysis. A novel murine model that produces embryonic stem cell-derived vessels will be used, and innovative technology will be developed to analyze vessel formation in explants of tetraploid embryos and transgenic mice as well.
The specific aims are: 1) to determine how endothelial cell division and blood vessel morphogenesis are coordinated in time and space; and 2) to determine if regulators that negatively modulate or block the cell cycle affect blood vessel morphogenesis. Blood vessels carrying endothelial-specific GFP localized to both the membrane and nuclear DNA will be used for dynamic image analysis to provide a baseline set of relationships. Then cell cycle regulators will be inappropriately expressed in endothelial cells, and effects on morphogenesis will be evaluated using dynamic imaging. These manipulations will be done with both wild-type vessels and mutant vessels formed from cells lacking the vascular endothelial growth factor (VEGF) receptor flt-1. This work should provide a strong framework to begin to understand how cell division and morphogenesis are integrated during blood vessel formation, information critical to reconstituting the process for therapeutic uses.
