Extra domain-B containing fibronectin (EDB(+) FN), a recently proposed marker of angiogenesis, has been shown to be expressed in a number of human cancers and in ocular neovascularization in patients with proliferative diabetic retinopathy. To gain molecular understanding of the functional significance of EDB(+) FN, we have investigated possible regulatory mechanisms of induction and its role in endothelial cell proliferation and angiogenesis. Human vascular endothelial cells were cultured in high levels of glucose, and fibrogenic growth factors, transforming growth factor-beta1 (TGF-beta1) and endothelin-1 (ET-1). Our results show that high glucose levels, TGF-beta1, and ET-1 upregulated EDB(+) FN expression. Treatment of cells exposed to high glucose with TGF-beta1 neutralizing antibody and ET receptor antagonist prevented high glucose-induced EDB(+) FN expression. In order to elucidate the functional significance of EDB(+) FN upregulation, cells were subjected to in vitro proliferation and angiogenesis assays following EDB peptide treatment and specific EDB(+) FN gene silencing. Our results show that exposure of cells to EDB peptide increased vascular endothelial growth factor (VEGF) expression, endothelial proliferation, and tube formation. Furthermore, specific EDB(+) FN gene silencing prevented both basal and high glucose-induced VEGF expression and reduced the proliferative capacity of endothelial cells. In conclusion, these results indicate that EDB(+) FN is involved in endothelial cell proliferation and vascular morphogenesis, findings which may provide novel avenues for the development of anti-angiogenic therapies.

Fibroblast growth factor-2 (FGF-2) has been implicated in vascular smooth muscle cell (SMC) migration, a key process in vascular disease. We demonstrate here that FGF-2 promotes SMC motility by altering beta1 integrin-mediated interactions with the extracellular matrix (ECM). FGF-2 significantly increased surface expression of alpha2beta1, alpha3beta1, and alpha5beta1 integrins on human SMCs, as assessed by flow cytometry. The greatest increase was for the collagen-binding alpha2beta1 integrin. Despite this, FGF-2 did not increase SMC adhesion to type I collagen but instead promoted SMC elongation and SMC motility. The latter was evaluated by using a microchemotaxis chamber and by digital time-lapse video microscopy. Although FGF-2 was not chemotactic for human SMCs, cells preincubated with FGF-2 displayed a 3.1-fold increase in migration to the undersurface of porous type I collagen-coated membranes and a 2.1-fold increase in migration speed on collagen. Furthermore, chemotaxis to platelet-derived growth factor-BB on collagen was significantly greater in SMCs exposed to FGF-2. FGF-2-induced elongation and migration on collagen were inhibited by a blocking anti-alpha2beta1 antibody; however, SMC adhesion to collagen was unaffected. SMC migration on fibronectin was also enhanced by FGF-2, although less prominently: migration through porous membranes increased 1.8-fold, and migration speed increased 1.3-fold. Also, FGF-2 completely disassembled the smooth muscle alpha-actin-containing stress fiber network contemporaneously with the change in integrin expression and cell shape. We conclude that (1) exogenous FGF-2 promotes SMC migration and potentiates chemotaxis to PDGF-BB; (2) the promigratory effect of FGF-2 is especially prominent on type I collagen and is mediated by upregulation of alpha2beta1 integrin; and (3) FGF-2 disassembles actin stress fibers, which may promote differential utilization of alpha2beta1 integrin for motility but not adhesion. This dynamic SMC-ECM interplay may be an important mechanism by which FGF-2 facilitates SMC motility in vivo.

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