Unit of General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, University of Brescia, 25123 Brescia; 
*Institute of Human Anatomy, Histology and Embryology,° Department of Biomedical Sciences and Human Oncology, University of Bari, 70124 Bari; 
§Biosearch Italia S.p.A., 21040 Gerenzano, Varese, Italy.

The fibrinolytic system is thought to play a central role in angiogenesis as indicated by the capacity of various cytokines and angiogenic growth factors, including fibroblast growth factor-2 (FGF2), to upregulate urokinase-type plasminogen activator (uPA) and/or its receptor in endothelial cells of different origin. 

In turn, in vitro experimental evidences suggest that proteolytic degradation of the extracellular matrix (ECM) by activation of the uPA/plasmin system may affect growth factor activity and bioavailability, even though no direct in vivo observations are available to support this hypothesis. 

Here we demonstrate that fetal bovine aortic endothelial GM 7373 cells overexpressing human uPA (uPA-R5 cells) are able to release ¹²⁵I-FGF2 bound to endothelial ECM in a plasmin-dependent manner. 

Accordingly, uPA-R5 cells exert a potent angiogenic activity in vivo when applied on the top of the chorioallantoic membrane (CAM) of the chick embryo. In contrast, mock-transfected Neo2 cells are unable to release ECM-bound ¹²⁵I-FGF2 and are poorly angiogenic in the CAM assay. Neovascularization elicited by uPA-R5 cells is significantly reduced by neutralizing anti-FGF2 antibodies to values similar to those observed in Neo2 cell-treated CAMs. 

In keeping with these observations, purified human uPA stimulates neovascularization of the CAM in the absence of an inflammatory response. Also in this case, the angiogenic activity of purified uPA was inhibited by neutralizing anti-FGF2 antibodies. Phenylmethylsulfonyl fluoride-treated uPA and the non-catalytic, receptor-binding amino-terminal fragment of uPA were instead non angiogenic in the CAM assay. 

Taken together, the data indicate that uPA is able to induce angiogenesis in vivo in the chick embryo CAM via a plasmin-dependent degradation of ECM that causes the mobilization of stored endogenous FGF2.
 
 
 

J. Cell Sci. (1999) 112:4213-4221