Vol. zy 22, No. zy 2 April 1998 Alcohol-Induced Upregulation of Activators and Fibrinolytic Activity in Endothelial Cells Plasminogen Cultured Human Michael zyxwvutsrq L. Aikens, Hernan E. Grenett, Raymond L. Benza, Edlue M. Tabengwa, Glenda C. Davis, and Francois M. Booyse Clinical studies suggest that moderate alcohol consumption may decrease the risk for coronary artery disease and myocardial infarc- tion. This effect may be attributed, in part, to the alcohol-mediated increase in endothelial cell (EC)-mediated fibrinolytic activity medi- ated by the increase in synthesis and/or activity of tissue-type plas- minogen activators (t-PAS) and/or urokinase-type PA (u-PAS). To de- termine whether low alcohol levels (0.01 to zyxwvuts 0.1%, v/v) induced the expression of these proteins, cultured human saphenous vein ECs (HSVECs)were preincubated in the absence/presence of ethanol for 5 to 120 min at 37"C, washed, refed, and further incubatedfor 8 and 24 hr without alcohol. PA mRNA (reverse transcriptase-polymerase chain reaction) and secreted antigen (ELSA) levels were analyzed after incubation for 8 and 24 hr and the net expression zyxwvut of (sustained) endogenous PA-mediated surface-localized HSVEC fibrinolytic ac- tivity (plasmin generation)quantitated by activation of '%Glu-plas- minogen after incubation for 24 hr. A brief 5 to 30 min preincubation (induction) of both t-PA and U-PA antigen increased -3-fold (t-PA control, 14.2 zyxwvutsrqpo 2 1.7, plus alcohol, 25.4 2 5 ng/ml; u-PA control, 15 zyxwvu 2 0.8, plus alcohol, 46.4 2 1.3 ng/ml) and mRNA levels -2-fold, as compared with controls. Increased PA expression was associated with a significant concomitant -2-fold increase in surface-localized fibrinolytic activity (control, 96 2 2.8, plus alcohol, 255 2 42 fmol/ well). These combined results indicate that a brief exposure (<30 min) to low levels of alcohol can induce synthesis of EC-produced t-PA and u-PA resulting in an increased expression of HSVEC sur- face-localized fibrinolytic activity and may account, in part, for the apparent cardioprotective benefit associated with moderate alcohol consumption. Key Words: Fibrinolysis, Alcohol, PlasminogenActivators, Coro- nary Artery Disease. NUMBER of prospective cohort studies have demon- A strated a U-shaped relationship between alcohol con- sumption and overall mortality.'-4 The positive association between moderate alcohol consumption and a reduced overall mortality is due largely to a reduced risk of cardio- vascular Most epidemiological and experi- mental studies of moderate alcohol intake and coronary artery disease (CAD) have focused on alcohol-induced changes in the lipid profile, including favorable increases in high-density lipoprotein-cholesterol (HDL-C).536 However, From the Division of Cardiovascular Disease, Department of Medicine, Received for publication June 26, 1997; accepted October 7, 1997 M.L.A. zyxwvutsrqpo was supported by the Institutional National Research Services Reprint requests: Michael L. Aikens, M.D., 845 19th Street South, BBRB Copyright zyxwvutsrqpon 0 1998 by The Research Society on Alcoholism. University of Alabama at Birmingham, Birmingham, Alabama. Award T.32 HL-07703 during the course of this study. 809, University of Alabama at Birmingham, Birmingham, A L 35294-2170. Alcohol Clin Exp Res. Vol 22, No 2, 1998: pp 375-381 differences in HDL-C may explain only 40% of the reduced risk.7 The other 60% reduced risk, and association may be mediated through the effects of alcohol on hemostasis. Normal hemostasis is maintained through a delicate bal- ance between coagulation and fibrinolytic proteins. Endo- thelial cells (ECs) play a major role in thromboregulation via their expression of surface-localized fibrinolytic activity and synthesis of fibrinolytic proteins, tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), and plasminogen activator inhibitor-type I (PAI-l), as well as their respective binding or receptor proteins.8 The net expression of EC surface-localized fibrinolytic activity in- volves the complex multicomponent interactions of these proteins and cell-bound plasminogen (Pmg) that serves to both regulate and localize the generation of fibrinolytic activity (plasmin generation) to the EC surface. Alcohol may alter the levels, activity, and interactions of fibrinolytic proteins and shift the hemostatic balance on the EC surface toward increased fibrinolytic activity, thus decreasing the vaso-occlusive events commonly associated with CAD and myocardial infarction (MI). Recent studies have supported this supposition as first evidenced by a cohort study of 87,526 moderate drinking nurses who were found to have a lower risk of both acute coronary events and ischemic stroke, but an increase risk of hemorrhagic stroke, suggest- ing that perturbation of fibrinolytic components may be involved.' Ridker et al.7 recently demonstrated a strong positive association between alcohol and t-PA antigen lev- els in 631 healthy men participating in the Physician Health Study, grouped according to amounts consumed. Several studies using various cell culture systems have shown that alcohol can induce protein expression through specific sig- nal transduction,'0-'2 binding to membrane regulatory pro- teins,11~13-15 or transcriptional/posttranscriptional regula- tory rne~hanisms.'~.'~ However, studies on the effects of alcohol on the expression of fibrinolytic proteins and activ- ity, in vitro are quite limited and incomplete. Laug18 de- scribed the enhancement of PA secretion in different cul- tured bovine EC types, after incubation with 0.5% alcohol at 37T, using a 1251-labeled fibrin-coated plate assay. Al- cohol has also been shown to increase t-PA secretion in a human melanoma cell line, known to produce t-PA.I9 We have recently described the rapid (<20 min) and direct effect of low alcohol (<0.1%) on the sustained increased 375