Mechanisms by which fibroblast growth factors stimulate DNA synthesis in coronary venular endothelial cells : a dissertation

"Submitted to the Office of Graduate Studies of Texas A & M University in partial fulfillment of the requirements for the degree of Doctor of Philosophy December 1992."Coronary angiogenesis, the formation of new microvessels in the heart, depends upon proliferation and migration of coronary venular endothelial cells (CVEC). We examined the molecular interactions of the angiogenic polypeptides, acidic and basic fibroblast growth factors (aFGF and bFGF, respectively) with cultured venular endothelial cells isolated from bovine heart. [^125]I-aFGF bound to a single class of sites while [^125]I-bFGF bound to both low-affinity heparin-like sites and high-affinity cell surface receptor sites. A 110-130 kDa cell surface FGF receptor protein was identified by crosslinking. aFGF and bFGF bound the same membrane receptor on CVEC. Heparin at 0.1 to 10 μg/ml potentiated aFGF, but not bFGF-induced proliferation and cell binding. At 37°C, bFGF rapidly bound to its receptor, internalized and was processed by two kinetically and biochemically distinguishable pathways. Up to 40-50% of total internalized bFGF was translocated to the nuclei of quiescent cells. Nuclear-bound [^125]I-bFGF showed little degradation even after 24 h, whereas cytoplasmic [^125]I-bFGF showed increased degradation to smaller fragments with time. Nuclear binding of bFGF reached equilibrium by 8 h, just before initiation of DNA synthesis, which began 9-12 h after growth factor addition. Other cell lines that expressed FGF receptors also bound, internalized, and showed nuclear accumulation of bFGF similar to CVEC, thus nuclear translocation of bFGF was not peculiar to CVEC. Two tyrosine kinase inhibitors, genistein and methyl 2,5- dihydroxycinnamate, showed reversible, dose-dependent inhibition of bFGF-stimulated DNA synthesis in CVEC with IC[50]'s of 12 and 3 μM, respectively. bFGF stimulated doseand time-dependent increases in tyrosine phosphorylation of CVEC proteins including the FGF receptor. Both compounds also exhibited dose-dependent inhibition of tyrosine phosphorylation of intracellular substrates induced by bFGF. A 2 hour pretreatment of quiescent CVEC with genistein blocked nuclear translocation but not cytoplasmic internalization of bFGF while the same treatment with methyl 2,5-dihydroxycinnamate inhibited both processes. FGF receptor tyrosine kinase activity appears to play a role nuclear translocation of bFGF and initiation of DNA synthesis in endothelial cells