Endothelial cell migration is critical to normal neovascularization, which is in turn essential for retinal development and function. Conversely, abnormal angiogenesis is the primary cause in the vast majority of diseases that result in vision loss, including age-related macular degeneration and diabetic retinopathy. While there has been some progress in understanding the factors that promote retinal angiogenesis, the mechanisms that drive the underlying endothelial cell migration process are not well understood. The research outlined here will develop a novel direct correlation approach to mechanistically dissect the cell locomotion functions required for endothelial migration both in vitro and in vivo. As a test case, this proposal will investigate the role of cofilin, an actin binding protein known to be important for motility in other cell types.
In Aim 1, the function of cofilin in endothelial cell migration in vitro will be examined by expression of cofilin mutants and RNA interference. Quantitative fluorescence image analysis and live-cell, time-lapse microscopy will be used to determine how the actin dynamics are altered in cells with perturbed cofilin function, and how this affects cell migration behavior.
In Aim 2, the functions of cofilin will be similarly perturbed in endothelial-precursor stem cells, which will then be intravitreally injected into neonatal mice. The ability of these stem cells to migrate during retinal neovascularization, and to integrate into the retinal vasculature will be monitored by quantitative fluorescence microscopy analyses of the retinal ? vasculature at various time intervals. This novel integrated approach will lead to a fundamental ? understanding of the endothelial cell migration processes that are specifically required for retinal ? neovascularization, and will lay the groundwork for future comprehensive studies of regulatory ? networks controlling retinal angiogenesis. Furthermore, this research will initiate development of in ? vitro assays with greater predictive power for identification of novel targets and compounds for ? anti-angiogenesis treatments. ? ? ?
| Nowak, Roberta B; Fischer, Robert S; Zoltoski, Rebecca K et al. (2009) Tropomodulin1 is required for membrane skeleton organization and hexagonal geometry of fiber cells in the mouse lens. J Cell Biol 186:915-28 |
| Weber, Kari L; Fischer, Robert S; Fowler, Velia M (2007) Tmod3 regulates polarized epithelial cell morphology. J Cell Sci 120:3625-32 |
| Fischer, Robert S; Yarmola, Elena G; Weber, Kari L et al. (2006) Tropomodulin 3 binds to actin monomers. J Biol Chem 281:36454-65 |
