STIMULATION OF PAI-1 EXPRESSION IN ENDOTHELIAL-CELLS BY CULTURED VASCULAR SMOOTH-MUSCLE CELLS

Regulation of endothelial cell (EC) plasminogen activator inhibitor ty pe-1 (PAI-1), the primary physiological inhibitor of tissue-type plasm inogen activator (TPA) and urokinase-type plasminogen activator (UPA), by various stimuli has been well characterized. We report the upregul ation 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 condit ions as determined by enzyme-linked immunosorbent assay. Cocultured hu man umbilical vein ECs and SMCs, or human umbilical artery ECs and SMC s, displayed a 73% and 68% increase, respectively, in released PAI-1. SMC-derived stimulatory factor release showed tissue specificity, sinc e only human aortic, umbilical vein, and umbilical artery SMCs upregul ated PAI-1 synthesis, whereas SMCs from human mammary artery, pulmonar y artery, and saphenous vein did not. Stimulation of EC PAI-1 by SMC C M was both time and concentration dependent, with as much as five- and fourfold increases in supernatants and lysates, respectively. PAI-1 s ynthesis and activity in ECs from other vascular beds were also upregu lated by SMC CM. Northern blot analysis paralleled the protein results , showing as much as a 2.7-fold increase in specific EC PAI-1 mRNA exp ression after incubation with SMC CM for 8 hours. PAI-1 stimulatory ac tivity in SMC CM was completely abolished by boiling or incubation wit h protamine sulfate and was reduced by transient acidification or hepa rin-Sepharose pretreatment by 33% or 48%, respectively. The stimulator y factor(s) appeared to have a molecular mass of 23 kD as determined b y gel filtration. Heat and acid lability precluded transforming growth factor-beta involvement. SMC CM also proved negative for interleukin- 1 alpha activity, tumor necrosis factor-alpha activity, and basic fibr oblast growth factor antigen. These results suggest that a soluble fac tor(s) secreted constitutively by SMCs is probably distinct from trans forming growth factor-beta, interleukin-1 alpha, tumor necrosis factor -alpha, and basic fibroblast growth factor and may influence intravasc ular fibrinolysis through regulation of PAI-1 gene expression.