Evaluation of 14-1B mutant of recombinant human plasminogen activator inhibitor type 1 (PAI-1) as a potential anti-angiogenic and anti-cancer agent

The process of new blood vessel formation from pre-existing blood vessels, collectively known as angiogenesis, is stimulated both under normal physiological and pathological conditions. In the past 30 years it was discovered that tumors are able to stimulate angiogenesis. During tumor cell-stimulated angiogenesis, a network of blood vessels is formed through which nutrients and oxygen are delivered to the tumor and the by-products of metabolism are removed. In addition, the newly formed vessels are very often used by tumors as a route of metastasis. Evidence has accumulated that the u-PA/plasmin system is important in the process of angiogenesis and it is activated during initial stages of this process. The u-PA/plasmin system is responsible for generating of increased local proteolytic activity that helps to create a space for newly developing capillaries. In addition, the u-PA/plasmin system is responsible for the modulation of the adhesive properties of endothelial cells to the extracellular matrix and cell-cell interactions. The u-PA is a serine protease that is responsible for generation of plasmin, a broad-substrate specific proteolytic enzyme. Numerous studies showed that u-PA is over-expressed in many types of human cancers. Binding of u-PA to its receptor, u-PAR, confines the proteolytic activity of urokinase to the cell surface. Many studies shown that inhibition of u-PA proteolytic activity effectively inhibited the invasive and metastatic potential of tumor cells.; In the present study we investigated whether the inhibition of u-PA bound to its receptor expressed on the surface of the stimulated endothelial cells using recombinant 14-1B PAI-1 (T_1/2 = 130.7 hours) would slow down or inhibit the invasion of stimulated endothelial cells into the surrounding extracellular matrix and prevent formation of new blood vessels. Our results showed that recombinant 14-1B PAI-1 is a potent anti-angiogenic agent and that exogenous delivery of this inhibitor prevented formation of new blood vessels as investigated using chicken chorioallantoic membrane. In addition, 14-1B PAI-1 inhibited sprout formation by aggregates of endothelial cells suspended in fibrin gels. Recombinant 14-1B PAI-1 was shown to have anti-cancer properties and inhibited the growth of LnCaP prostate tumors in SCID mice.