Distribution of Smooth Muscle Cell Derived Urokinase in Cocultures with Endothelial Cells

In this study we report evidence for a significant contribution of smooth muscle cells (SMC) to the fibrinolytic potential of the vascular wall. SMC produce urokinase type plasminogen activator (uPA) which is predominantly deposited into the extracellular matrix (ECM) and secreted into the abluminal compartment, in a system which uses human amniotic membrane as growth support. SMC cultivated on amniotic membranes furthermore display a passage dependent uPA secretion pattern. Early passages (e.g. passage #5) secrete 116 ± 6.5 ng uPA/24 h into the conditioned media whereas in supernatants of late passages (e.g. passage #10) no uPA is detectable. These late passage SMC, however, appear to primarily secrete uPA into the amniotic membrane. Comparison of membrane extracts from early and late passages applying fibrin autography reveals a switch from predominantly free (i.e. non complexed) fibrinolytic activity (M r of about 54 000) in early passages to a mainly inhibitor complexed (= inactive) form (M r of about 100 000) in late passages. De novo synthesis of uPA and deposition into the membrane is demonstrated by 35 S-methionine labeling of SMC. Immunoabsorption analysis of membrane extracts with monoclonal antibodies against uPA suggests the immunological identity of one of the major labeled protein bands deposited by SMC into the membranes with uPA. Cocultivation of SMC with HUVEC on either side of human amniotic membranes results in an increase of uPA secretion into the lower (SMC) compartment, representing the abluminal side of this vessel wall model, from 63 ± 2.9 to 165±38.2ng uPA/24 h. Incubation of SMC grown on gelatine coated plastic dishes with only the conditioned media of HUVEC, however, does not change uPA secretion of SMC, whereas the incubation of HUVEC with the SMC conditioned media results in a significant decrease of uPA added with the SMC conditioned media exogenously (from 148 ± 20.3 to 87 ± 9.5 ng uPA/24 h) suggesting an uptake and/or degradation of uPA by HUVEC. These data indicate that the mechanism of uPA induction in the coculture system requires the simultaneous presence of both cell types; furthermore it appears that endothelial cells might contribute to the polar secretion pattern of uPA by uptake and/or degradation of uPA secreted into the luminal compartment.