Increased expression of plasminogen activator inhibitor-1 (PAI-1) mRNA in atherosclerotic human arteries suggests a linkage between PAI-1 gene expression and cellular proliferation, the fundamental feature of atherosclerosis. To investigate whether smooth muscle cell (SMC) proliferation influences overall fibrinolytic properties of the vascular wall, we examined the effect of serial in vitro passaging of human SMCs on tissue plasminogen activator (TPA) and PAI-1 synthesis levels as well as the ability to modulate TPA and PAI-1 synthesis of human umbilical vein endothelial cells (HUVECs). As in vivo correlates for such late-passage cells in culture, SMCs derived from human atherosclerotic plaques were used, because they are thought to have already undergone numerous cell doublings. We observed an increase of PAI-1 secretion (from 591 +/- 106 to 2952 +/- 290 ng PAI-1.10(5) cells-1.24 h-1) with a concomitant fourfold to fivefold increase of PAI-1 mRNA levels, as well as a decrease of TPA secretion (from 118 +/- 34 to 8 +/- 1.3 ng TPA.10(5) cells-1.24 h-1) and a twofold to threefold decrease of TPA mRNA levels with increasing in vitro passage number (from passage 3 to 11) of normal pulmonary artery smooth muscle cells (PASMCs) (P < .05). SMCs derived from atherosclerotic plaques of coronary arteries (CASMCs) displayed higher levels of PAI-1 antigen synthesis (3093 +/- 507 ng PAI-1.10(5) cells-1.24 h-1) with an approximately twofold increase of PAI-1 mRNA levels, as well as decreased levels of TPA antigen synthesis (10 +/- 1.6 ng TPA.10(5) cells-1.24 h-1) with an approximately 1.5- to 2-fold decrease of TPA mRNA levels in passage 1, compared with their counterparts derived from normal-appearing arterial tissue of the same vessel (1794 +/- 525 ng PAI-1.10(5) cells-1.24 h-1; 17 +/- 5 ng TPA.10(5) cells-1.24 h-1) (P < .001; P < .01). Incubation of HUVEC cultures with the 24-hour conditioned media (CM) of early-passage PASMCs decreased endothelial PAI-1 antigen synthesis by approximately 42% (P < .001) and endothelial PAI-1 mRNA levels about twofold to threefold (P < .001), whereas by incubation with the 24-hour CM of late-passage PASMCs, endothelial PAI-1 antigen synthesis was upregulated by 68% (P = .001), with a concomitant twofold increase of endothelial PAI-1 mRNA levels (P < .001). The apparent MW of this heat- and acid-stable PAI-1 upregulating factor appears to be between 50 and 100 kD, as judged by ultrafiltration. Incubation of HUVEC cultures with the 24-hour CM of early-passage CASMCs derived from normal-appearing arterial tissue showed no significant influence on endothelial PAI-1 synthesis, whereas incubation with late-passage normal CASMCs, as well as early-passage atherosclerotic CASMCs from the same vessel, increased endothelial PAI-1 antigen secretion by 45% and 48% (P < .001), with a concomitant 1.5 fold to 2-fold increase of endothelial PAI-1 mRNA levels (P < .05). No significant change in endothelial TPA synthesis was observed by incubation with CM of either PASMCs (early or late passage) or CASMCs (atherosclerotic or normal). These data suggest that SMC proliferation is associated with (1) increased SMC PAI-1 synthesis as well as decreased TPA synthesis and (2) upregulation of endothelial PAI-1 synthesis by SMC CM. This phenomenon is observed with either late passages of normal PASMCs and CASMCs or early passages of atherosclerotic plaque CASMCs. This suggests that proliferating SMCs are a major regulator of the fibrinolytic potential within the vessel wall, thereby contributing to the thrombotic risk associated with the development of atherosclerosis.