Plasminogen activator inhibitor type-1 mutants regulate angiogenesis of human umbilical and lung vascular endothelial cells

Abstract

Plasminogen activator inhibitor type-1 (PAI-1) is considered one of the key regulators of tumor invasion, metastasis, as well as cancer-related angiogenesis. The literature suggests that PAI-1 plays a dual role in these processes because it inhibits plasmin-originated proteolysis and binds to vitronectin or integrins. Stimulation or inhibition of angiogenesis largely depends on which of these elements PAI-1 interacts. Wild PAI-1 converts quickly into its latent, inactive form and loses its anti-proteolytic activity, but still binds to vitronectin and integrins. Thus we constructed PAI-1s with extended half-life to prolong their anti-proteolytic activity. We have analyzed the effects of sprout formation inhibition by PAI-1s on two functionally different endothelial cell (EC) systems, human umbilical vein endothelial cells (HUVEC), expressing moderate amounts of urokinase (uPA), and human lung microvascular endothelial cells (HLMVEC), expressing high amounts of this enzyme. We have used wild-type PAI-1 (wPAI-1) (t(1/2) = 1.6 h) and PAI-1 cysteine mutants (CysPAI-1) characterized by their prolonged half-life time (hDbetaT) (t(1/2) = 63.6 h and t(1/2) = 7,000 h). We have observed a significant inhibitory dose-dependent effect exerted by the CysPAI-1s on sprout formation by HUVEC and HLMVEC cells. The inhibition rate was considerably stronger in lung capillary cell cultures and significantly more pronounced for CysPAI-1 mutants with longer anti-uPA activity (betaT). wPAI-1 with a short anti-proteolytic half-life has induced sprout formation in HUVEC, but not in HLMVEC cultures. This difference in behavior was most likely related to the presence of excessive amounts of uPA in HLMVEC cells and the known mechanism of clearing PAI-1/uPA/uPAR complexes from the cell surface. A less efficient system of HUVEC cells might give wPAI-1 the chance to interact with non-proteolytic pathways of angiogenesis stimulation. We conclude that while the anti-proteolytic properties of PAI-1 constructs are preserved, these proteins inhibit angiogenesis and inhibitory activity dominates over any stimulatory effects of PAI-1.