Effect on PAI - 1 Synthesis by ECs and SMCs in Coculture PAI - 1 , ng / 16 h Percent Increase

Regulation of endothelial cell (EC) plasminogen activator inhibitor type-1 (PAI-1), the primary physiological inhibitor of tissue-type plasminogen activator (TPA) and urokinase-type plasminogen activator (UPA), by various stimuli has been well characterized. We report the upregulation of secreted and intracellular PAI-1 in human umbilical ECs when cocultured with human smooth muscle cells (SMCs) on amniotic membranes or incubated with SMC conditioned medium (CM) under serum-free conditions as determined by enzymelinked immunosorbent assay. Cocultured human umbilical vein ECs and SMCs, or human umbilical artery ECs and SMCs, displayed a 73% and 68% increase, respectively, in released PAI-1. SMC-derived stimulatory factor release showed tissue specificity, since only human aortic, umbilical vein, and umbilical artery SMCs upregulated PAI-1 synthesis, whereas SMCs from human mammary artery, pulmonary artery, and saphenous vein did not. Stimulation of EC PAI-1 by SMC CM was both time and concentration dependent, with as much as fiveand fourfold increases in supernatants and lysates, respectively. PAI-1 synthesis and activity in ECs from other vascular beds were also upregulated by SMC CM. Initiation of fibrinolysis occurs with the generation of the active serine protease plasmin by tissue-type (TPA) and urokinase-type (UPA) plasminogen activators. In blood, plasminogen activator inhibitor type-1 (PAI-1) is the major physiological inhibitor of plasminogen activation; PAI-1 is tightly complexed with TPA and is thought to prevent fibrin clot dissolution. PAI-1 also inhibits UPA and thus plays a pivotal role in cell migration, neoplasia, angiogenesis, and tissue remodeling in addition to intravascular clot formation and dissolution. Because net fibrinolytic activity reflects the balance between plasminogen activators and plasminogen activator inhibitors, identification of the factors involved in the regulation of PAI-1 is important. In cultured endothelial cells (ECs), PAI-1 is upregulated by various stimuli, including dexamethasone, thrombin, lipopolysaccharide, tumor necrosis factor-a (TNF-a), interleukin-1 (IL-1), transforming growth factor-/? (TGF-/3), basic fibroblast growth factor (bFGF), and lipoprotein(a).^ Received May 17, 1993; revision accepted February 7, 1994. From the Department of Diagnostic Haematology, Royal Melbourne Hospital, and the Department of Medicine, University of Melbourne, Melbourne, Victoria, Australia. Correspondence to Marisa Gallicchio, Department of Medicine, Monash University, Monash Medical Centre, 246 Clayton Rd, Melbourne, Victoria 3168, Australia. Northern blot analysis paralleled the protein results, showing as much as a 2.7-fold increase in specific EC PAI-1 mRNA expression after incubation with SMC CM for 8 hours. PAI-1 stimulatory activity in SMC CM was completely abolished by boiling or incubation with protamine sulfate and was reduced by transient acidification or heparin-Sepharose pretreatment by 33% or 48%, respectively. The stimulatory factor(s) appeared to have a molecular mass of 23 kD as determined by gel filtration. Heat and acid lability precluded transforming growth factor-/!! involvement. SMC CM also proved negative for interleukin-la activity, tumor necrosis factor-a activity, and basic fibroblast growth factor antigen. These results suggest that a soluble factor(s) secreted constitutively by SMCs is probably distinct from transforming growth factor-^, interleukin-la, tumor necrosis factor-a, and basic fibroblast growth factor and may influence intravascular fibrinolysis through regulation of PAI-1 gene expression. (Arterioscler Thromb. 1994;14:815-823.)