Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are angiogenic molecules whose combined mitogenic activity is potently synergistic. However, the molecular mechanism underlying this synergy is incompletely understood. We examined whether VEGF and bFGF affect expression of each other or alter expression of the VEGF receptor KDR in retinal capillary endothelial cells. In addition, we investigated the intracellular signaling mechanisms involved in this response. VEGF-induced [3H]thymidine uptake was tightly correlated with KDR mRNA and protein concentrations, suggesting that increased KDR expression might account for VEGF's synergistic activity in the presence of bFGF. bFGF (10 ng/ml) induced KDR mRNA expression within 4 h and attained a 4.0-fold increase after 24 h. KDR protein expression was increased 7.5-fold after 48 h. VEGF (= 50 ng/ml) did not alter bFGF, VEGF, or KDR mRNA expression under serum-deprived conditions. In contrast, VEGF increased KDR mRNA expression 87% under growth conditions and 2.9-fold under serum-deprived conditions in the presence of bFGF. The protein kinase C (PKC) agonist phorbol myristate acetate (PMA) induced KDR mRNA expression 5.1-fold at 100 nmol/l. bFGF increased p44/p42 mitogen-activated protein kinase (MAPK) phosphorylation within 5 min, reaching a maximum within 15 min and remaining significantly elevated for >6 h. bFGF-induced MAPK phosphorylation and KDR mRNA expression were almost completely inhibited by 5 micromol/l GFX, a non-isoform-selective PKC inhibitor. MAPK inhibitor PD98059 reduced KDR mRNA expression 72% at concentrations that inhibited bFGF-induced MAPK phosphorylation 100%, suggesting that pathways in addition to MAPK might also be involved. Inhibitors of the beta isoform of PKC (LY333531), protein kinase A (PKA) (H89), and phosphotidylinositol (PI) 3 kinase (wortmannin) had no significant effect. These data suggest that bFGF stimulates KDR expression through a PKC and p44/p42 MAPK-dependent pathway not primarily involving the beta isoform of PKC, PKA, or PI-3 kinase. Since bFGF induces VEGF expression and since increased KDR expression potentiates VEGF action, resulting in additional KDR expression and marked mitogenic activity, these data provide a novel mechanistic explanation for the angiogenic synergy between VEGF and bFGF.

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