Craig Lundgren

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.

Increased intramural expression of plasminogen activator inhibitor type 1 after balloon injury: a potential progenitor of restenosis.

OBJECTIVES This study was performed to determine whether altered gene expression of plasminogen activator inhibitor type 1 (PAI-1) occurs within the arterial wall after experimentally induced balloon injury. BACKGROUND PAI-1, known to inhibit fibrinolysis in the circulation and to be present within atherosclerotic vessels, may influence proteolysis in the arterial wall and neointimal formation after angioplasty. METHODS In rabbit carotid arteries subjected to balloon injury, both PAI-1 gene and protein expression were assayed sequentially with the use of Northern blotting, in situ hybridization and immunohistochemical studies. RESULTS In uninjured, normal vessels PAI-1 messenger ribonucleic acid (mRNA) was not detectable by Northern blotting or in situ hybridization. However, injury was followed within 3 h by increases in PAI-1 mRNA (3.2 kb) of 5.9-fold compared with that in contralateral control carotid arteries (Northern blots). PAI-1 mRNA was detectable by in situ hybridization early after injury first in adventitia; after 24 h it was particularly prominent in the media. From 1 to 4 weeks after injury it was consistently detectable and was localized in neointimal vascular smooth muscle and endothelial cells at a time when neointimal thickening was marked. Cells of both types exhibited PAI-1 protein detected immunohistochemically. In vessels maintained in organ culture after balloon injury in vivo, sustained increases in PAI-1 activity appeared in conditioned media as well. CONCLUSIONS Our results indicate that balloon injury simulating angioplasty in patients induces intramural expression of PAI-1 in vascular smooth muscle and endothelial cells. The decreased cell surface fibrinolytic activity likely to result from the increased PAI-1 expression may initiate or exacerbate mural thrombosis. Accordingly, excessive stimulation with clot-associated mitogens may stimulate vascular smooth muscle cell proliferation, which, coupled with increased accumulation of extracellular matrix attributable to decreased plasmin-mediated degradation, may contribute to restenosis.

Modulation of expression of monocyte/macrophage plasminogen activator activity and its implications for attenuation of vasculopathy.

BACKGROUND The binding of urokinase-type plasminogen activator (uPA) to its receptor (uPAR) on cell surfaces has the potential to influence degradation of extracellular matrix (ECM). Thus, uPA bound to monocyte/macrophages and its interactions with plasminogen activator inhibitors types 1 and 2 (PAI-1 and PAI-2) may modify atherogenesis by altering cell-associated proteolytic activity, degradation of ECM, and neointimal formation at sites of vascular injury. METHODS AND RESULTS To determine whether the expression of proteins on the surface of cells involved in fibrinolysis changes in human cells in response to mediators implicated in atherogenesis, we exposed U937 cells (an immortal human monocyte-like cell line) to transforming growth factor-beta (TGF-beta) and to thrombin. Induction of uPAR mRNA occurred with TGF-beta (5 ng/mL) in a time-dependent fashion (P = .05; n = 4). Thrombin (5 National Institutes of Health [NIH] U/mL) increased uPAR mRNA by 2.8-fold above control (n = 4) without altering PAI-1 mRNA or protein synthesis (n = 4). The increase in uPAR gene expression in cells exposed to either TGF-beta or thrombin translated into a functional increase in cell-surface proteolytic activity. Under control conditions, U937 cells expressed PAI-2 but not PAI-1 mRNA. PAI-2 mRNA expression increased (P < .05; n = 4) with thrombin (5 NIH U/mL) but was suppressed by TGF-beta (5 ng/mL). TGF-beta induced PAI-1 mRNA within 6 hours accompanied by a 9-fold increase in PAI-1 protein from 6 hours (2.9 +/- 1.9 ng/mL) to 24 hours (20.0 +/- 9.6 ng/mL, P = .005; n = 3) paralleled by increased synthesis as shown in metabolic labeling experiments with 35S-methionine and immunoprecipitation of labeled PAI-1. PAI-1 mRNA and protein expression were seen in human coronary artery atherectomy specimens as well and were localized to analogous monocyte/macrophages and to smooth muscle cells as judged from results of in situ hybridization and immunocytochemistry studies. CONCLUSIONS The results indicate that there is induction of PAI-1 and uPAR in U937 cells exposed to TGF-beta and thrombin. In atheroma, analogous processes may modulate early migration of luminal monocytes into the subintimal space and proteolysis of ECM. Thus, cell surface, monocyte-directed fibrinolysis may influence atherosclerosis, restenosis, or both.

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.

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.

A Vitronectin Receptor Antagonist Inhibits Neointimal Formation After Balloon Arterial Injury in Rabbits in Vivo

The authors have previously shown that cell-matrix interactions mediated via the matrix protein ligand vitronectin derived from serum and its cell surface αVβ3 integrin receptor regulate migration of human aortic smooth muscle cells (SMC) in vitro. Arginine-glycine- aspartic acid (RGD) sequence of vitronectin (residues 45 to 47) is the critical domain for interactions. In this study the authors sought to determine whether interference with vitronectin-receptor interaction in the arterial wall would alter development of neoin timal hyperplasia in a rabbit model. After balloon-induced injury of the carotid artery (2 fr Fogarty, three times) vitronectin was observed in the intima and upper third of the media (immunostaining, n= 3). In 6 rabbits the vitronectin receptor antagonist cyclic RGD peptide (1 mM) was locally applied to the injured artery by use of ethylene vinyl acetate copolymers (25%). RAD peptide (1 mM, n=6) and ethylene vinyl acetate copolymer without peptide (n=6) served as an inactive control. At twenty-eight days rabbits were killed, carotid arteries were fixed, and mean intima and media area was determined by computer-assisted planimetry. (continued on next page) (Abstract continued) The mean intima/media ratio in control balloon-injured arteries without peptide was 0.43 ±0.08 (SD). In injured RAD peptide-treated arteries the ratio was 0.39 ±0.09 (P= NS). In injured, cyclic RGD peptide-treated arteries the intima/media ration was 0.14 ±0.02 (P < 0.05). There was no significant difference in media area in the two groups. These results suggest that (1) vitronectin-vitronectin receptor interaction mediates SMC migration in vivo, and (2) localized application of a vitronectin receptor antagonist to the arterial wall after balloon injury modifies this process and results in a significant reduction in the development of neointimal hyperplasia. Vitronectin-vitronectin receptor interaction may be an additional attractive target for pharmacologic manipulations aimed at limiting restenosis after vascular injury.