Steven L. Brown

Elaboration of Type-1 Plasminogen Activator Inhibitor From Adipocytes A Potential Pathogenetic Link Between Obesity and Cardiovascular Disease

BACKGROUND Obesity is known to predispose to attenuated fibrinolysis attributable to increased concentrations in plasma of type-1 plasminogen activator inhibitor (PAI-1), the primary physiological inhibitor of endogenous fibrinolysis. PAI-1 is present in neointimal vascular smooth muscle cells and lipid-laden macrophages. METHODS AND RESULTS The present study was designed to determine whether PAI-1 expression occurs in adipose tissue as well, thereby potentially contributing to increased cardiovascular risk associated with obesity. 3T3-L1 preadipocytes were differentiated into adipocytes by exposing them to isobutylxanthine (0.5 mmol/L) and dexamethasone (0.25 mumol/L) over 7 days and incubated for 24 hours with transforming growth factor-beta (TGF-beta), known to augment PAI-1 synthesis in several cell types and to be released from platelets when they are activated. TGF-beta increased PAI-1 activity in the conditioned media of the 3T3-L1-derived cells in a concentration-dependent fashion without significantly affecting cell proliferation. Western blotting and immunoprecipitation of 35S-labeled PAI-1 showed that the increased PAI-1 activity paralleled increased PAI-1 protein. Northern blotting showed that increased PAI-1 mRNA preceded increased accumulation of PAI-1 activity and protein in the conditioned media. Furthermore, TGF-beta (10 ng/g body wt) administered in vivo increased PAI-1 activity in mouse plasma and PAI-1 mRNA expression in mouse adipose tissue. CONCLUSIONS Increased plasma PAI-1 activity in obese human subjects may result from PAI-1 release from an increased mass of adipose tissue, particularly in association with thrombosis and elaboration of TGF-beta from platelet alpha-granules into the circulation. The increased PAI-1 may exacerbate vascular disease by shifting the balance between thrombosis and thrombolysis toward thrombosis and consequently exposing luminal surfaces of vessels to mitogens associated with microthrombi over protracted intervals.

Attenuation of the Synthesis of Plasminogen Activator Inhibitor Type 1 by Niacin A Potential Link Between Lipid Lowering and Fibrinolysis

BACKGROUND Plasminogen activator inhibitor type 1 (PAI-1), the primary physiological inhibitor of endogenous plasminogen activators, has been implicated as a potentiating factor in atherogenesis as well as in coronary thrombosis. We and others have observed attenuation of PAI-1 expression by gemfibrozil both in vivo and in vitro. METHODS AND RESULTS To determine whether other lipid-lowering agents with different mechanisms of action exert similar effects, we exposed Hep G2 cells, a highly differentiated human hepatoma cell line, to selected concentrations of niacin. Accumulation of PAI-1 protein, assayed with an ELISA, decreased in conditioned media by 72% in 48 hours in a specific, concentration-dependent fashion. Metabolic labeling experiments demonstrated a decrease in the rate of PAI-1 synthesis. Northern blot analysis demonstrated a preceding, parallel, and specific decrease in the concentration of PAI-1 mRNA. Niacin attenuated the increased PAI-1 synthesis induced by mediators released from thrombi as well. Thus, with 4.25 ng/mL transforming growth factor-beta 1, PAI-1 accumulation increased 4.5-fold in conditioned media in 48 hours. However, niacin attenuated the increase by 65%. Again, both the rate of PAI-1 synthesis and PAI-1 mRNA were reduced. The increased plasma PAI-1 activity and PAI-1 mRNA in liver induced by dexamethasone (0.8 mg IP) in vivo in rats were attenuated by 3 weeks of pretreatment with niacin. CONCLUSIONS These results suggest that niacin, by decreasing PAI-1 expression, may potentiate fibrinolysis, thereby decreasing the stimulation of atherogenesis by clot-associated mitogens associated with microthrombi. Furthermore, the results imply that a pathogenetic link may exist between intracellular lipid metabolism and regulation of expression of fibrinolytic system components.

Dependence of Human Vascular Cell Surface Proteolysis on Expression of the Urokinase Receptor

To delineate the role of binding of urokinase type plasminogen activator (uPA) to its receptor (uPAR) in the local generation of plasmin by endothelium, we transfected spontaneously transformed immortalized human vascular endothelial cells that express high levels of uPA but low levels of uPAR with human uPAR complementary DNA. Compared with nontransfected cell, the stably transformed clonal cell line exhibited (a) a > 10-fold increase in steady-state uPAR mRNA levels documented with Northern blot analysis (n = 3), (b) a 2.8-fold increase in cell surface expression of uPAR protein quantified by enzyme linked immunosorbent assay (n = 3), (c) a 2.9-fold increase in specific binding of radiolabeled single chain uPA (n = 4), and (d) markedly increased matrix adhesion. The participation of uPAR in cell surface proteolysis was apparent based on a 3.0-fold increase in cell associated plasmin activity (n = 3) and a 2.3-fold increase in lysis of noncrosslinked fibrin clots (n = 5). Thus, local generation of plasmin and consequent degradation of fibrin are likely to be promoted by cell surface localization of uPA by uPAR in cellular constituents of the vessel wall. Furthermore, genetic engineering of endothelium to enhance expression of uPAR may confer resistance to thrombosis or restenosis associated with endovascular stents.

Effect of etofibrate and nicanartine on plasminogen activator inhibitor type-1 production in vitro by cultured vascular cells and on plasma plasminogen activator inhibitor type-1 activity in vivo in rabbits

Abstract The effect of etofibrate and nicanartine on plasminogen activator inhibitor type-1 (PAI-1) production in vitro and on plasma PAI-1 activity in vivo was determined. Both agents decreased baseline and transforming growth factor-β induced—PAI-1 synthesis in cultured human vascular endothelial and smooth muscle cells and attenuated the increase in plasma PAI-1 activity and aortic PAI-1 gene expression induced by hypercholesterolemia and mechanically induced vascular injury in rabbits in vivo. Attenuation of the increase in plasma PAI-1 activity and aortic PAI-1 gene expression was associated with the decrease in the aortic atherosclerotic lesion area. Etofibrate and nicanartine may be useful in ameliorating the atherosclerotic process by directly acting on PAI-1 synthesis in vascular cells.