The general aim of this project is to better define the functional effects of bFGF on vascular endothelial cells.
Specific aim 1 addresses the issue of an autocrine or intracrine role of bFGF in vascular cells. The argument is made that while a great deal is known about bFGF distribution in tissues, and the biology of exogenous bFGF is well-known, no information is available regarding the physiological role of endogenous bFGF. The principal investigator wishes to address the issue of the role of endogenous bFGF by evaluating several parameters of cultured endothelial cells including survival, migration, and proliferation.
Specific aim 2 is to characterize the general regulation of bFGF protein and mRNA synthesis in this endothelial cell preparation. Initial studies will include systematic investigation of mRNA levels in vascular cells as a function of their growth state. At various time points after plating of vascular cells, total mRNA will be isolated, and probed for bFGF mRNA as well as bFGF protein levels under these various conditions. Following these baseline studies, the regulation of bFGF expression and content will be evaluated following induction of bFGF release by sublethal injury, as well as during increased bFGF mRNA biosynthesis induced by TGFb without increased bFGF level. In the latter experiment, the assessment will be made about whether mRNA is translated and the overall bFGF protein turnover time is increased to maintain steady state levels or whether the mRNA is not translated.
Specific aim 3 assesses the effect of bFGF on gene expression in vascular endothelial cells. The thrust of these experiments is to find the spectrum of genes that might be regulated by bFGF. By providing exogenous bFGF, attempts will be made to examine the changes in gene expression induced by bFGF. This series of experiments will use the differential display analysis and cloning techniques to specifically identify genes that are activated following bFGF stimulation in primary endothelial cell cultures. The outcome of these studies is to identify novel genes that are regulated in endothelial cells by bFGF.
| Wagner, J A; D'Amore, P A (1986) Neurite outgrowth induced by an endothelial cell mitogen isolated from retina. J Cell Biol 103:1363-7 |
