UROKINASE-DEPENDENT ANGIOGENESIS IN-VITRO AND DIACYLGLYCEROL PRODUCTION ARE BLOCKED BY ANTISENSE OLIGONUCLEOTIDES AGAINST THE UROKINASE RECEPTOR

The plasminogen activator system is known to play a crucial role in th e angiogenesis process by modulating the adhesive properties of endoth elial cells to the extracellular matrix and cell-cell interaction. In the present study, we demonstrated that the urokinase-type plasminogen activator (u-PA) induced neovascular growth in the avascular rabbit c ornea and dose-dependently promoted growth, chemotaxis, and matrix inv asion of cultured endothelial cells. Interaction between u-PA and its receptor appears to be mandatory for the angiogenic effect of u-PA bec ause monoclonal antibodies anti-u-PA and anti-u-PA receptor (u-PAR) bl ocked the proangiogenic effects of u-PA at the endothelial cell level. We then assessed the signaling pathway activated in endothelial cells by u-PA. u-PAR activation by u-PA produced de novo synthesis of diacy lglycerol (DAG) from glucose by a cytochalasin B-inhibitable mechanism , indicating the involvement of a specific glucose transporter (GLUT). Endothelial cells expressed GLUT2, whose activation was tyrosine kina se-dependent and protein kinase C (PKC)-independent. The increase of g lucose uptake led to DAG production, which resulted in PKC activation/ translocation. Impairment of u-PAR availability by monoclonal antibodi es and by antisense oligonucleotides (aODN) against u-PAR mRNA inhibit ed glucose uptake, DAG neosynthesis, and PKC activation, resulting in the blockade of endothelial cell proliferation, chemotaxis, and chemoi nvasion. These data suggest that u-PAR activation consequent to the bi nding of u-PA can be regarded as an ''angiogenic switch'' and disclose the possibility that an anti-u-PAR aODN strategy may efficiently targ et endothelial cell function to control angiogenesis in vivo.