ANTIFIBRINOLYTIC PROPERTIES OF THE VASCULAR WALL - DEPENDENCE ON THE HISTORY OF SMOOTH-MUSCLE CELL DOUBLINGS IN-VITRO AND IN-VIVO

Increased expression of plasminogen activator inhibitor-1 (PAI-1) mRNA in atherosclerotic human arteries suggests a linkage between PAI-1 ge ne expression and cellular proliferation, the fundamental feature of a therosclerosis. To investigate whether smooth muscle cell (SMC) prolif eration influences overall fibrinolytic properties of the vascular wal l, we examined the effect of serial in vitro passaging of human SMCs o n tissue plasminogen activator (TPA) and PAI-1 synthesis levels as wel l as the ability to modulate TPA and PAI-1 synthesis of human umbilica l vein endothelial cells (HUVECs). As in vivo correlates for such late -passage cells in culture, SMCs derived from human atherosclerotic pla ques were used, because they are thought to have already undergone num erous 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 con comitant fourfold to fivefold increase of PAI-1 mRNA levels, as well a s a decrease of TPA secretion (from 118+/-34 to 8+/-1.3 ng TPA.10(5) c ells(-1).24 h(-1)) and a twofold to threefold decrease of TPA mRNA lev els with increasing in vitro passage number (from passage 3 to 11) of normal pulmonary artery smooth muscle cells (PASMCs) (P<.05). SMCs der ived from atherosclerotic plaques of coronary arteries (CASMCs) displa yed 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 approximate to 1.5- to 2-fold decrease of TPA mRNA levels in passage 1, compared with their c ounterparts 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 decrea sed endothelial PAI-1 antigen synthesis by approximate to 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, en dothelial PAI-1 antigen synthesis was upregulated by 68% (P=.001), wit h 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 ultrafiltrat ion. Incubation of HUVEC cultures with the 24-hour CM of early-passage CASMCs derived from normal-appearing arterial tissue showed no signif icant influence on endothelial PAI-1 synthesis, whereas incubation wit h late-passage normal CASMCs, as well as early-passage atherosclerotic CASMCs from the same vessel, increased endothelial PAI-1 antigen secr etion by 45% and 48% (P<.001), with a concomitant 1.5-fold to 2-fold i ncrease of endothelial PAI-1 mRNA levels (P<.05). No significant chang e in endothelial TPA synthesis was observed by incubation with CM of e ither PASMCs (early or late passage) or CASMCs (atherosclerotic or nor mal). 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 phenom enon is observed with either late passages of normal PASMCs and CASMCs or early passages of atherosclerotic plaque CASMCs. This suggests tha t proliferating SMCs are a major regulator of the fibrinolytic potenti al within the vessel wall, thereby contributing to the thrombotic risk associated with the development of atherosclerosis.