Urokinase Receptor-Dependent Upregulation of Smooth Muscle Cell Adhesion to Vitronectin by Urokinase

Abstract

The plasminogen activator system has been implicated in the modulation of the response to vascular injury. Although urokinase-type plasminogen activator (uPA) and its receptor (uPAR) may enhance matrix degradation as well as migration and invasion by smooth muscle cells (SMCs), their roles in cell adhesion are uncertain. Therefore, we examined the ability of uPA and uPAR to modulate adhesion of cultured human vascular SMCs to various matrices. We demonstrated a dose-dependent stimulation of adhesion by single-chain uPA (scuPA) to vitronectin (maximum 1.55-fold [+/-0. 04-fold] increase, 10 nmol/L, P<0.002) but not to laminin, collagen I, or collagen IV. Baseline adhesion to vitronectin was completely inhibited by both EDTA and RGD peptide but was restored to >40% of control in the presence of scuPA (P=0.001 and 0.046, respectively). Adhesion to vitronectin was also significantly enhanced by the amino-terminal fragment of uPA (P=0.007) and two-chain, high-molecular-weight uPA (P<0.01) but not by the low-molecular-weight fragment of uPA, which lacks the receptor-binding domain. Aprotinin, a plasmin inhibitor, had no effect on baseline or scuPA-stimulated adhesion, suggesting a plasmin-independent process. Preincubation of scuPA with soluble uPAR inhibited scuPA stimulation of adhesion by 88+/-14% (P=0.01), as did pretreatment of SMCs with phosphatidylinositol-specific phospholipase C, which removes glycophosphatidylinositol-anchored proteins, including uPAR. Antibodies to both alphavbeta3 and alphavbeta5 integrin inhibited baseline adhesion but not scuPA stimulation. Finally, coating plates with scuPA alone enabled cell adhesion, which could be inhibited by both soluble uPAR and anti-uPAR antibodies. These data suggest that uPA stimulates adhesion of SMCs specifically to vitronectin and that it is mediated by an interaction with uPAR. Upregulation of both proteins after vascular injury may facilitate migration through stimulation of both matrix degradation and cell adhesion.

Eptifibatide and 7E3, but Not Tirofiban, Inhibit α v β 3 Integrin–Mediated Binding of Smooth Muscle Cells to Thrombospondin and Prothrombin

“…These results are consistent with previous studies of human aortic 21 and human iliac 22 SMCs in which 7E3 at concentrations of 20 and 60 g/mL had no effect on adhesion to vitronectin. Similarly, neither 7E3 nor eptifibatide had any effect on HASMC attachment to collagen-or fibronectin-coated or noncoated tissue culture plates ( Figure 3B).…”

Novel Bi- and Trifunctional Inhibitors of Tumor-Associated Proteolytic Systems

“…It has been demonstrated that interaction of GPI-uPAR with αvβ3 integrin is essential for uPAR-dependent adhesion to vitronectin and disruption of the uPAR/integrin complexes by uPAR-binding peptides (p25, M25, and α325) blocks adhesion to vitronectin (Wei et al, , 1999Simon et al, 2000;Pluijm et al, 2001). uPA and ATF have been shown to enhance uPAR/vitronectin binding affinity and, by this, increase cell adhesion to Vn (Wei et al, 1994Kanse et al, 1996;Stahl and Mueller, 1997;Chang et al 1998;Yebra et al, 1999). It is possible that the interaction of the uPAR binding site-harboring trifunctional inhibitor, with uPAR will also enhance uPAR/vitronectin binding affinity and by this promote cell adhesion.…”

Deficiency of Urokinase-Type Plasminogen Activator–Mediated Plasmin Generation Impairs Vascular Remodeling During Hypoxia-Induced Pulmonary Hypertension in Mice

“…32 Hypoxia-induced pulmonary vascular remodeling was assessed by two different methods. 34 First, the peripheral vessel density (defined as the number of vessels per 100 alveoli) was determined. Peripheral arteries were defined as all vessels landmarked to airway structures distal to the terminal bronchioli.…”