Thomas K. Nordt

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.

Attenuated Fibrinolysis and Accelerated Atherogenesis in Type II Diabetic Patients

Patients with hyperinsulinemia, defined by increased concentrations of IRI in plasma, experience increased cardiovascular mortality. In type II diabetic patients, the increase in IRI may reflect, in part, not only insulin but also proinsulinemia as a result of impaired conversion of proinsulin to insulin by pancreatic β-cells. High IRI is accompanied by attenuation of endogenous fibrinolytic activity and increased plasma PAI-1, the primary physiological inhibitor of t-PA. Concordant increases of plasma PAI-1 and plasma IRI appear to reflect direct effects of insulin and proinsulin on the synthesis and secretion of PAI-1 by endothelial and liver cells as judged from results of studies in vitro. Because attenuated fibrinolysis may predispose to thrombosis, the increased exposure of luminal surfaces of vessels to atherogenic, clot-associated mitogens and chemoattractants may activate macrophages and potentiate proliferation of vascular smooth muscle cells. Accordingly, increased concentrations of plasma IRI may contribute to macrovascular disease in diabetic patients by Impairing endogenous fibrinolysis.

Augmentation of the synthesis of plasminogen activator inhibitor type-1 by precursors of insulin. A potential risk factor for vascular disease.

BACKGROUND Both vascular disease and elevated concentrations in plasma of plasminogen activator inhibitor type-1 (PAI-1) are prominent in patients with non-insulin-dependent diabetes mellitus (NIDDM). We and others have hypothesized that the increased PAI-1 may contribute to acceleration of atherosclerosis in this condition and in other states characterized by insulin resistance as well. Surprisingly, however, elevations of PAI-1 decrease when type II diabetic patients are treated with exogenous insulin, as do circulating concentrations of the precursor of insulin, proinsulin, in plasma. Accordingly, the increased PAI-1 in patients with NIDDM may reflect effects of precursors of insulin rather than or in addition to those of insulin itself. To assess this possibility directly, this study was performed to identify potential direct effects of proinsulin and proinsulin split products on synthesis of PAI-1 in liver cells, thought to be the major source of circulating PAI-1 in vivo. METHODS AND RESULTS Hep G2 cells (highly differentiated human hepatoma cells) were exposed to human proinsulin, des(31,32)proinsulin and des(64,65)proinsulin (split products of proinsulin), or C-peptide. Accumulation of PAI-1 in conditioned media increased in a time- and concentration-dependent fashion in response to the two des-intermediates [3.3-fold with des(31,32)proinsulin and 4.5-fold with des(64,65)proinsulin]. C-peptide elicited no increase. Stimulation was transduced at least in part by the insulin receptor as shown by inhibition of stimulation by insulin receptor antibodies, mediated at the level of PAI-1 gene expression as shown by the 2.2- to 2.9-fold increases in steady-state concentrations of PAI-1 mRNA, and indicative of newly synthesized protein as shown by results in metabolic labeling experiments. CONCLUSIONS Our results are consistent with the hypothesis that precursors of insulin (proinsulin and proinsulin split products), known to be present in relatively high concentrations in plasma in patients with NIDDM and conditions characterized by insulin resistance, may directly stimulate PAI-1 synthesis, thereby attenuating fibrinolysis and accelerating atherogenesis.

Stimulation by Proinsulin of Expression of Plasminogen Activator Inhibitor Type-I in Endothelial Cells

In patients with non-insulin-dependent diabetes mellitus, concentrations in plasma of insulin and its precursors, proinsulin and split proinsulin, are increased. Because increased concentrations of plasminogen activator inhibitor type-1 (PAI-1) occur also, we hypothesized that proinsulin and split proinsulin may augment endothelial cell PAI-1 expression, thereby potentially attenuating endogenous fibrinolysis and accelerating atherosclerosis. Proinsulin increased PAI-1 activity in conditioned media of endothelial cells as did split proinsulin, paralleled by increased expression of PAI-1 mRNA. These effects of proinsulin were not dependent on its conversion to insulin nor on its interactions with the insulin receptor. The proinsulin stimulation of PAI-1 expression was not attenuated by either anti-insulin receptor antibodies or a 100-fold excess of insulin. Furthermore, proinsulin-mediated increases in PAI-1 expression were not inhibited by a 500-fold excess of insulinlike growth factor I. In addition, inhibition of tyrosine kinase, which mediates many of the diverse effects of insulin and insulinlike growth factor I, did not attenuate the effect of proinsulin. These results indicate that proinsulin augments PAI-1 expression, potentially contributing to vasculopathy in patients with non-insulin-dependent diabetes mellitus.

Augmentation of synthesis of plasminogen activator inhibitor type-1 in arterial endothelial cells by glucose and its implications for local fibrinolysis.

Because of the frequent occurrence of premature cardiovascular disease in patients with non-insulin-dependent, type II diabetes mellitus (NIDDM), the attenuated fibrinolytic activity of plasma from type II diabetic patients with increased concentrations of plasminogen activator inhibitor type-1 (PAI-1), and the fact that insulin stimulates synthesis of PAI-1 by human hepatic cells in vitro, we and others have hypothesized that accelerated vascular disease in type II diabetes may result in part from impaired fibrinolysis secondary to excessive elaboration of PAI-1 stimulated by insulin. Alternatively, the hyperglycemia associated with type II diabetes could influence the synthesis and secretion of PAI-1 directly. The present study was performed to determine whether PAI-1 secretion is or is not sensitive to the prevailing concentration of glucose in the conditioned medium of endothelial and liver cells, which are thought to be the major sources of circulating PAI-1 in vivo. Confluent cells were exposed to 0, 2.8, 5.6, 11.1, or 22.2 mmol/L (0, 50, 100, 200, or 400 mg/dL) glucose in medium without serum and subsequently to media with or without insulin (7.3 nmol/L). Secretion of PAI-1 by highly differentiated human hepatoma (Hep G2) cells did not increase as a function of increasing concentrations of glucose, whether or not insulin was present. In contrast, with pig aortic endothelial cells, the secretion of PAI-1 increased significantly with extracellular glucose with or without insulin. The increases in PAI-1 were specific (as shown by metabolic labeling experiments) and not attributable to osmotic effects (as shown by replacement of glucose by sorbitol).(ABSTRACT TRUNCATED AT 250 WORDS)

Direct attenuation of plasminogen activator inhibitor type-1 expression in human adipose tissue by thiazolidinediones.

Adipose tissue produces substantial amounts of plasminogen activator inhibitor type-1 (PAI-1), an established cardiovascular risk factor. This study evaluated PAI-1 expression in human adipose tissue in response to thiazolidinediones, insulin sensitising drugs activating peroxisome proliferator-activated receptor-gamma (PPAR-gamma). Troglitazone, rosiglitazone, and ciglitazone significantly reduced PAI-1 protein expression in human preadipocytes under basal conditions and after stimulation of the cells with TGF-beta. Pioglitazone had no effect. In human adipocytes all four thiazolidinediones significantly attenuated PAI-1 expression. Signalling appeared to be mediated via PPAR-gamma and effects reflected, at least in part, changes in transcription. Accordingly, patients with insulin resistance may benefit from treatment with thiazolidinediones with respect to diminution of PAI-1 expression in adipose tissue and consequent potential reduction of cardiovascular risk.

Flow Cytometric Monitoring of Glycoprotein IIb/IIIa Blockade and Platelet Function in Patients With Acute Myocardial Infarction Receiving Reteplase, Abciximab, and Ticlopidine Continuous Platelet Inhibition by the Combination of Abciximab and Ticlopidine

BackgroundImprovement of thrombolysis may be achieved by concomitant strong platelet inhibition. To monitor platelet function in patients with myocardial infarction (n=46) who were treated with the fibrinolytic agent reteplase, the glycoprotein (GP) IIb/IIIa blocker abciximab, and the ADP receptor antagonist ticlopidine, we developed a flow cytometric assay. Methods and ResultsBinding of abciximab to platelets was directly monitored as the percentage of platelets stained by a goat anti-mouse antibody. Blood drawn 10 minutes and 2 hours after the start of therapy with reteplase and abciximab and during the 12-hour infusion of abciximab demonstrated a maximal blockade of GP IIb/IIIa (10 minutes, 86.2±10.3%; 12 hours, 85.8±7.1%). Starting at 24 hours, abciximab binding gradually decreased (24 hours, 74.6±16.2%; 48 hours, 66.8±14.9%; 72 hours, 60.5±16.7%; 96 hours, 49.4±17.8%; 120 hours, 35.8±16.4%; and 144 hours, 29.9±15.3%). Binding of a chicken anti-fibrinogen antibody to platelets, indicating the level of functional blockade of GP IIb/IIIa, was inversely correlated with the binding of abciximab (r =−0.72, P <0.0001). In blood drawn at 10 minutes, platelet aggregation was maximally inhibited but recovered within 48 hours even if the majority of GP IIb/IIIa receptors were still blocked by abciximab. Reteplase did not influence abciximab binding and did not activate platelets, as measured by P-selectin expression, fibrinogen binding, and platelet aggregation. Platelet inhibition that was achieved during the first 24 hours by abciximab was directly maintained by additional treatment with ticlopidine. ConclusionsFlow cytometric monitoring of platelet function allows differentiation of the effects of reteplase, abciximab, and ticlopidine. The combination of abciximab and ticlopidine is an attractive therapeutic strategy that provides a fast and continuous platelet inhibition.

Constitutive biosynthesis of plasminogen activator inhibitor type-1 (PAI-1) by cultured human aortic endothelial cells independent of insulin.

BackgroundBoth tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1) are synthesized by vascular endothelium, whereas hepatocytes synthesize PAI-1 but not t-PA. Non-insulin-dependent diabetes mellitus (NIDDM) is associated with decreased fibrinolytic activity in blood secondary to increased PAI-1 activity, and the increase in PAI-1 activity is correlated with the magnitude of elevation of plasma immunoreactive insulin. To determine whether the increased PAI-1, known to be associated with accelerated coronary artery disease in non-diabetic subjects, is a consequence of direct effects of insulin on endothelial cells, we performed the present study with primary cultures of human aortic endothelial cells. MethodsEndothelial cells isolated from human aortas from donor hearts for transplantation were grown to confluence and exposed to selected concentrations of agonists. Accumulation of t-PA and PAI-1 in conditioned media was quantified, as was PAI-1 activity. ResultsInsulin at pharmacologic concentrations did not alter either PAI-1 or t-PA production by the human aortic endothelial cells, although insulin stimulated PAI-1 synthesis in human hepatoma (Hep G2) cells as expected. Transforming growth factor-β (TGF-β) stimulated endothelial cell PAI-1 production markedly, indicating that the cells could respond positively to stimulation in vitro. PAI-1 activity in the conditioned media was zero under all conditions, which was indicative of the rapid inactivation and degradation of PAI-1 known to occur in media devoid of vitronectin. ConclusionsThe decreased fibrinolytic activity in blood seen in patients with NIDDM appears to reflect direct effects of insulin or its precursor on hepatocytes rather than on endothelial cells

Inhibition by fibrates of plasminogen activator inhibitor type-1 expression in human adipocytes and preadipocytes

Plasminogen activator inhibitor type-1 (PAI-1), an established marker and mediator of cardiovascular risk, is produced extensively in adipose tissue. Fibrates are hypolipidemic peroxisome proliferator activated receptor-alpha (PPARalpha) agonists. Recent laboratory and clinical observations indicate that they are also anti-atherosclerotic. Mechanisms responsible, however, remain to be fully understood. The present study was designed to elucidate modulation of PAI-1 expression in adipose cells by fibrates as a potential mechanism. Expression of PPARalpha was verified by PCR, immunohistochemistry, and Western blotting. In cultured preadipocytes and adipocytes gemfibrozil and fenofibrate significantly reduced PAI-1 protein expression by up to 55 +/- 5% and 34 +/- 4% under basal conditions and up to 56 +/- 6% and 31 +/- 6% under conditions of stimulation of the cells with 40 pM transforming growth factor (TGF)beta, respectively. Quantification of mRNA showed that the gemfibrozil-induced effect was at least in part regulated at the transcriptional level. Incubations with non-fibrate PPARalpha agonists showed similar reductions in PAI-1 expression. The decrease in PAI-1 expression induced by gemfibrozil was inhibited by MK886, a PPARalpha inhibitor. Furthermore, preadipocytes isolated from PPARalpha-deficient mice produced significantly more PAI-1 than those from wild-type mice upon stimulation with TGFbeta. Finally, fenofibrate reduced PAI-1 expression both in plasma and adipose tissue of hyperlipidemic mice. Our data support the view that PPARalpha activation down-regulates PAI-expression in adipose cells that may contribute in part to the reduction in cardiovascular mortality seen with fibrates in clinical trials.

Human Aortic Vascular Smooth Muscle Cells Digest Extracellular Matrix by Elaboration of Plasminogen Activators: Implications for Atherogenesis.

Migration and proliferation of vascular smooth muscle cells (SMCs) are hallmarks of atherogenesis and restenosis after angioplasty. Digestion of surrounding extracellular matrix (ECM) may be a critical link. To determine whether invasion of ECM by human aortic SMCs (HASMCs) depends on proteolytic digestion mediated by the cells themselves, we characterized ECM digestion in terms of solubilization of3H-proline-labeled ECM, produced by the use of rat aortic SMCs, by HASMCs under various conditions. Pasmin alone (10 μg/ml) digested 80% of ECM in 2 hours. HASMCs in 10% fetal bovine serum cultured on ECM that was not exposed to plasmin digested 48% of the ECM in 7 days. When HASMCs were cultured on plasmin-pretreated ECM, only 14% of the residual ECM was digested. Conditioned media or cells cultured on porous membrane 1 mm removed from the ECM had no effect. Baseline secretion of tissue-type plasminogen activator (t-PA) into the media by HASMCs averaged 3.9 ng/105 cells/24 hr and baseline secretion of type-1 plasminogen activator inhibitor (PAI-1) averaged 1300 ng/105 cells/24 hr. Thrombin (5 U/ml) increased t-PA antigen production by 184% without altering PAI-1 activity and increased ECM degradation by 43% in 7 days. Transforming growth factor-β (TGF-β) decreased t-PA antigen production, increased PAI-1 activity, and decreased ECM degradation. These results suggest that (1) HASMCs can digest naturally produced ECM; (2) plasminogen-dependent mechanisms requiring cell contact are important in the initiation of this phenomenon; and (3) thrombin in the vicinity of clots may modulate the fibrinolytic and proteolytic properties of SMC through t-PA after vascular injury.