Induction of Plasminogen Activator Inhibitor Type 1 and Type 1 Collagen Expression in Rat Cardiac Microvascular Endothelial Cells by Interleukin-1 and Its Dependence on Oxygen-Centered Free Radicals

Abstract

Background —Ischemia with or without reperfusion induces the release of diverse products from monocytes, including cytokines such as interleukin-1 (IL-1). To determine whether these phenomena modulate fibrinolysis and potentially exacerbate impairment of the macrocirculation, microcirculation, or both, we characterized the effects of IL-1 on the expression of fibrinolytic system and matrix proteins in rat cardiac microvascular endothelial cells (CMECs). Methods and Results —Confluent CMECs were exposed to IL-1 in serum-free medium for 24 hours, and cell-conditioned medium was assayed for plasminogen activator inhibitor type 1 (PAI-1), the primary physiological inhibitor of plasminogen activators, and for type 1 collagen with Western blotting. IL-1 (2 ng/mL) specifically increased the accumulation of PAI-1 (4.4±0.6-fold; mean±SD; n=9) without affecting tissue plasminogen activator (t-PA) or urokinase plasminogen activator (u-PA) levels, which remained unchanged. IL-1 increased the accumulation of collagen in conditioned media by 3.5±0.7-fold (n=6). Conversely, the accumulation of both PAI-1 and collagen induced by IL-1 was inhibited with an IL-1 receptor antagonist (200 ng/mL; n=6) and with cycloheximide (10 μg/mL; n=6), implying that protein synthesis was a requirement for the effect. To determine whether the IL-1 effect was mediated by induction of oxygen-centered free radical production, known to be induced by IL-1, we exposed the cells to the hydroxyl radical scavenger tetramethylthiourea (10 mmol/L) and observed abolition of the IL-1–induced increase in the expression of PAI-1 and collagen (n=6). Conversely, superoxides (generated with 10 mU/mL xanthine oxidase plus 0.6 mmol/L hypoxanthine, and 100 μmol/L hydrogen peroxide) induced the accumulation of PAI-1 and collagen (n=6). IL-1 (1 μg/kg body wt) and lipopolysaccharide (50 μg/kg body wt) administered in vivo increased PAI-1 protein in rat hearts as detected with Western blotting and PAI-1 immunostaining of rat heart microvessels, indicating the effects delineated in vitro were paralleled by effects in vivo. Conclusions —These results indicate that IL-1–induced oxygen-centered free radicals stimulate elaboration of PAI-1 and collagen by CMECs. Accordingly, microvascularly mediated inhibition of fibrinolysis may predispose to the persistence of microvascular thrombi, thereby contributing to impaired microcirculatory function, the no-reflow phenomenon, and cardiac dysfunction after ischemia and reperfusion.