Angiogenesis may play an important role in the physiologic response of the myocardium to ischemia and infarction. In addition, cell growth factors may be implicated in the pathophysiology of myocardial hypertrophy. The mitogenic protein, basic fibroblast growth factor (bFGF), is plentiful in the myocardium, and forms complexes in vivo with cell surface and extracellular matrix heparan sulfate molecules. Heparan sulfates may serve to protect bFGF from proteolytic degradation, maintain it in insoluble form until released to exert its actions, and possibly provide a vehicle for the transmembrane export of newly synthesized bFGF. Detailed structural information regarding specific bFGF-binding proteoglycans is limited, particularly in the heart. Such information may clarify the physiologic relationships between bFGF and heparan sulfate, which will in turn provide insight into the sequence of events leading to myocardial angiogenesis or hypertrophy.
The specific aims of the proposed studies are: 1. Obtain partial amino acid sequences from peptide fragments of the core protein from cardiac bFGF-binding proteoglycan. bFGF-binding proteoglycan will be purified from total ventricular plasma membrane using anion-exchange and bFGF-affinity chromatography. The core protein of this proteoglycan will be cleaved with cyanogen bromide, followed by partial sequencing of bFGF-binding peptide fragments. 2. Raise antibodies against portions of the cardiac bFGF-binding proteoglycan. Synthetic peptides and cyanogen bromide cleavage products will be used as the immunogen. Both approaches will be attempted. 3. Identify a cDNA clone coding for the peptide fragment(s) identified in Specific Aim 1. Two methods of screening are proposed. A bovine heart cDNA library will be screened using the antibody(s) raised in Specific Aim 2, as well as cognate oligonucleotide probes synthesized based on partial amino acid sequence data. Information obtained by these studies will lead to the determination of the primary structure of a bFGF-binding proteoglycan present on myocardial cell membranes. cDNA clones identified in Specific Aim 3 will be sequenced and the primary structure of the proteoglycan core protein deduced. Following the identification of a specific cardiac cell type producing the proteoglycan, cell-culture models will be developed which either under- or over-express the bFGF-binding ability of such a proteoglycan.
| Kilgore, J L; Timson, B F; Saunders, D K et al. (1994) Stress protein induction in skeletal muscle: comparison of laboratory models to naturally occurring hypertrophy. J Appl Physiol 76:598-601 |
| Ross, C R; Kubinak, S; Hale, C C (1993) Purification of a basic fibroblast growth factor-binding proteoglycan from bovine cardiac plasma membrane. Biochim Biophys Acta 1145:219-26 |
