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 the angiogenesis process by modulating the adhesive properties of endothelial 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 cornea and dose-dependently promoted growth, chemotaxis, and matrix invasion of cultured endothelial cells. Interaction between u-PA and its receptor appears to be mandatory for the angiogenic effect of u-PA because monoclonal antibodies anti-u-PA and anti-u-PA receptor (u-PAR) blocked 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 diacylglycerol (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 kinase-dependent and protein kinase C (PKC)-independent. The increase of glucose uptake led to DAG production, which resulted in PKC activation/translocation. Impairment of u-PAR availability by monoclonal antibodies and by antisense oligonucleotides (aODN) against u-PAR mRNA inhibited glucose uptake, DAG neosynthesis, and PKC activation, resulting in the blockade of endothelial cell proliferation, chemotaxis, and chemoinvasion. These data suggest that u-PAR activation consequent to the binding of u-PA can be regarded as an "angiogenic switch" and disclose the possibility that an anti-u-PAR aODN strategy may efficiently target endothelial cell function to control angiogenesis in vivo.
Istituto di Patologia Generale, Universita' di Firenze, Italy.
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G
Fibbi
R
R
Caldini
M
M
Chevanne
M
M
Pucci
N
N
Schiavone
L
L
Morbidelli
A
A
Parenti
H J
HJ
Granger
M
M
Del Rosso
M
M
Ziche
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D003094:Collagen / Q000187:drug effects; D003315:Cornea / Q000098:blood supply / Q000187:drug effects; D004075:Diglycerides / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D005109:Extracellular Matrix / Q000502:physiology; D051273:Glucose Transporter Type 2; D006801:Humans; D007797:Laminin / Q000187:drug effects; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects* / Q000502:physiology; D016376:Oligonucleotides, Antisense / Q000235:genetics* / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism; D011509:Proteoglycans / Q000187:drug effects; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*
D004075:Diglycerides; D004338:Drug Combinations; D051273:Glucose Transporter Type 2; D007797:Laminin; D009004:Monosaccharide Transport Proteins; D016376:Oligonucleotides, Antisense; C524898:PLAUR protein, human; D011509:Proteoglycans; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; C059485:matrigel; D003094:Collagen; D011493:Protein Kinase C; D014568:Urokinase-Type Plasminogen Activator