The long-term goal of this project is to understand how peptide growth factors regulate neovascularization in cardiovascular disease and cardiovascular development. An abnormality of the endothelium is a primary component of most major cardiovascular pathologies including atherosclerosis, myocardial infarction, stroke and hypertension. Likewise, growth regulation of the endothelium is critical in development because migrating endothelial cells establish the pattern for the embryonic vascular system. This project is part of a new program making use of transgenic mice and transgenic embryonic stem (ES) cells in gain and loss of function studies for basic fibroblast growth factor (FGF-2). FGF-2 is a prototypical peptide growth factor and angiogenic agent. The human FGF- 2 gene produces four protein isoforms (24,23,22,18 kD) from alternative translation start sites in its mRNA. The larger (24,23,22 kD) isoforms localize to the nucleus and the smaller isoform (18kD) is cytoplasmic and extracellular. We have made an FGF-2 transgenic overexpression mouse that produces all four human FGF-2 protein isoforms. The mouse has bone abnormalities, a cardiomyopathy, smooth muscle hyperplasia, and focal hyperplasia of the endothelium. Based on the cellular distribution and tissue specific expression patterns of the different FGF-2 isoforms that we found in the FGF-2 transgenic mouse, we hypothesize that they have alternative functions. To test this hypothesis we are making transgenic (gain of function or overexpression) mice and transgenic embryonic stem (ES) cells with mutant cDNA clones that selectively produce nuclear or cytoplasmic/extracellular FGF-2 isoforms. Concomitant loss of function experiments for FGF-2 by gene ablation in ES cells via homologous recombination are also proposed. Should our hypothesis prove correct, overexpression of specific FGF-2 isoforms in transgenic mice and embryonic stem (ES) cells will have different effects on neovascularization. We have outlined the following specific aims for this project: 1. Evaluate the effects of all four FGF-2 isoforms on pathological neovascularization and endothelial cell growth control. 2. Determine how individual isoforms of FGF-2 affect pathological neovascularization. 3. Develop an in vitro experimental system for neovascularization to study endothelial cell growth and differentiation using ES cell differentiation into embryoid bodies (EB). 4. Determine how loss of FGF-2 expression affects neovascularization by regulating endothelial cell growth and differentiation. Production of transgenic mice in this project serves two purposes, to experimentally test the functions of genes regulating neovascularization and to make animal models for cardiovascular diseases that can be used for therapeutics.
