Hesperetin and its sulfate and glucuronide metabolites inhibit TNF-α induced human aortic endothelial cell migration and decrease plasminogen activator inhibitor-1 (PAI-1) levels

Introduction: Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of mammalian plasminogen activators and an important regulator of cell migration. We have shown that tiplaxtinin, a small molecule, specific inhibitor of PAI-1, inhibits intimal hyperplasia in a murine vein graft model. However, little is known about the effects of pharmacological inhibition of PAI-1 on vascular cell migration under physiologically relevant conditions. Methods: We studied the effects of tiplaxtinin on migration of smooth muscle cells (SMCs) and endothelial cells (ECs). Results: Tiplaxtinin significantly inhibited migration of murine SMCs through 3-dimensional (3-D) collagen matrix in a concentration-dependent manner. Tiplaxtinin did not inhibit SMC proliferation, and it did not inhibit migration of PAI-1-deficient SMCs, suggesting that tiplaxtinin’s effect on SMCs was non-toxic and PAI-1-dependent. The anti-migratory effect of tiplaxtinin on SMCs was preserved in collagen 3-D matrix containing vitronectin and other extracellular matrix molecules, further supporting the physiological significance of the effect. In contrast to SMCs, tiplaxtinin did not inhibit migration of human aortic ECs in vitro or murine ECs in vivo, the latter assessed in a murine carotid injury model. To study the basis for the differential effect of tiplaxtinin on SMCs vs. ECs, we compared expression of LDL receptor-related protein 1 (LRP1), a motogenic receptor for PAI-1, between cell types by RT-PCR and found that LRP1 gene expression was significantly lower in ECs than in SMCs. Furthermore, recombinant PAI-1 stimulated the migration of wild-type mouse embryonic fibroblasts (MEFs), but not LRP1-deficient MEFs. Conclusions: Tiplaxtinin, a pharmacological inhibitor of PAI-1, inhibits SMC migration under physiological conditions, while having no inhibitory effect on EC migration. The differential effect of PAI-1 inhibition on SMCs vs. ECs appears to be mediated by LRP1 and may be of clinical significance, as it is advantageous to prevent intimal hyperplasia by inhibiting SMC migration without inhibiting EC migration, which is key to preserving an intact, anti-thrombotic vascular endothelium.

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TNF-α and insulin, alone and synergistically, induce plasminogen activator inhibitor-1 expression in adipocytes

AJP Cell Physiology ◽

10.1152/ajpcell.1999.276.6.c1391 ◽

1999 ◽

Vol 276 (6) ◽

pp. C1391-C1397 ◽

Cited By ~ 87

Author(s):

Tomohiro Sakamoto ◽

Janet Woodcock-Mitchell ◽

Kousuke Marutsuka ◽

John J. Mitchell ◽

Burton E. Sobel ◽

...

Keyword(s):

Plasminogen Activator ◽

Synergistic Effects ◽

Plasminogen Activator Inhibitor ◽

Plasminogen Activators ◽

System Capacity ◽

Plasminogen Activator Inhibitor 1 ◽

Tnf Α ◽

Factor Α ◽

Activator Inhibitor ◽

Pai 1

Obesity is associated with hyperinsulinemia and elevated concentrations of tumor necrosis factor-α (TNF-α) in adipose tissue. TNF-α has been implicated as an inducer of the synthesis of plasminogen activator inhibitor-1 (PAI-1), the primary physiological inhibitor of fibrinolysis, mediated by plasminogen activators in cultured adipocytes. To identify mechanism(s) through which TNF-α induces PAI-1, 3T3-L1 preadipocytes were differentiated into adipocytes and exposed to TNF-α for 24 h. TNF-α selectively increased the synthesis of PAI-1 without increasing activity of plasminogen activators. Both superoxide (generated by xanthine oxidase plus hypoxanthine) and hydrogen peroxide were potent inducers of PAI-1, and hydroxyl radical scavengers completely abolished the TNF-α induction of PAI-1. Exposure of adipocytes to TNF-α or insulin alone over 5 days increased PAI-1 production. These agonists exert synergistic effects. Results obtained suggest that TNF-α stimulates PAI-1 production by adipocytes, an effect potentiated by insulin, and that adipocyte generation of reactive oxygen centered radicals mediates the induction of PAI-1 production by TNF-α. Because induction of PAI-1 by TNF-α is potentiated synergistically by insulin, both agonists appear likely to contribute to the impairment of fibrinolytic system capacity typical in obese, hyperinsulinemic patients.

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Expression of plasminogen activator-inhibitor-1 (PAI-1) during cellular remodeling in proliferative glomerulonephritis in the rat.

Journal of Histochemistry & Cytochemistry ◽

10.1177/43.9.7642963 ◽

1995 ◽

Vol 43 (9) ◽

pp. 895-905 ◽

Cited By ~ 27

Author(s):

J L Barnes ◽

R J Mitchell ◽

E S Torres

Keyword(s):

Extracellular Matrix ◽

Cell Migration ◽

Plasminogen Activator ◽

Plasminogen Activator Inhibitor ◽

Proliferative Glomerulonephritis ◽

Plasminogen Activator Inhibitor 1 ◽

Glomerular Lesions ◽

Matrix Accumulation ◽

Activator Inhibitor ◽

Pai 1

Pericellular proteolysis involves the plasminogen activator/plasmin system and plays an important role in cell remodeling involving cell migration and extracellular matrix turnover. Studies in this laboratory have previously characterized a model of proliferative glomerulonephritis induced by Habu snake venom (HSV) in the rat that involves cell migration, proliferation, and extracellular matrix accumulation. Because plasminogen activator-inhibitor-1 (PAI-1) has been used as a marker for cell migration as well as matrix accumulation, we were interested in examining the temporal and spatial expression and cellular sources of PAI-1 mRNA and translated protein over the course of HSV-induced proliferative glomerulonephritis. The results showed a highly localized and progressive expression of PAI-1 mRNA and translated protein by in situ hybridization and immunohistochemistry at the margins and periphery of glomerular lesions 8 and 24 hr after HSV. The expression of PAI-1 in glomerular lesions localized to the same sites as mesangial cell marker proteins, desmin and Thy-1.1, indicating that mesangial cells synthesize this important regulator proteolysis. Few cells expressed PAI-1 in the central aspects of glomerular lesions at later time intervals (48 and 72 hr) when cell proliferation and expression of extracellular matrix (fibronectin protein and mRNA) were maximal. Therefore, the expression of PAI-1 in this model was associated more with early events related to cell migration than with proliferation or extracellular matrix synthesis. These observations support the hypothesis that the plasminogen activator/plasmin system is involved in cell migration in early remodeling during glomerular disease.

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The effect of endothelial cell overexpression of plasminogen activator inhibitor-1 on smooth muscle cell migration

Journal of Vascular Surgery ◽

10.1067/mva.2002.123687 ◽

2002 ◽

Vol 36 (1) ◽

pp. 164-171 ◽

Cited By ~ 12

Author(s):

Richard R. Proia ◽

Peter R. Nelson ◽

Mary Jo Mulligan-Kehoe ◽

Robert J. Wagner ◽

Arthur J. Kehas ◽

...

Keyword(s):

Smooth Muscle ◽

Cell Migration ◽

Endothelial Cell ◽

Smooth Muscle Cell ◽

Plasminogen Activator ◽

Muscle Cell ◽

Plasminogen Activator Inhibitor ◽

Plasminogen Activator Inhibitor 1 ◽

Smooth Muscle Cell Migration ◽

Activator Inhibitor

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Vascular Heme Oxygenase-1 Induction Suppresses Tissue Factor and Plasminogen Activator Inhibitor-1 Expressions

Blood ◽

10.1182/blood.v112.11.5447.5447 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5447-5447

Author(s):

Eriko Morishita ◽

Keiko Maruyama ◽

Akiko Sekiya ◽

Shigeki Ohtake ◽

Shinji Nakao ◽

...

Keyword(s):

Endothelial Cell ◽

Tissue Factor ◽

Plasminogen Activator ◽

Heme Oxygenase ◽

Plasminogen Activator Inhibitor ◽

Heme Oxygenase 1 ◽

Plasminogen Activator Inhibitor 1 ◽

Protein Levels ◽

Activator Inhibitor ◽

Pai 1

Abstract Objective - Heme oxygenase-1(HO-1), the rate-limiting enzyme of heme degradation, has recently been considered to have protective roles against various pathological conditions. 10 years have passed since we lost the first and the only patient of HO-1 deficiency. Since the patient of HO-1 deficiency showed endothelial cell injury and extremely enhanced coagulation and fibrinolytic parameters, we examined the effect of HO-1 modulation on tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) expression on endothelial cells. Methods and Results - Human umbilical vein endothelial cell (HUVEC) was stimulated with hemin (100mM), HO-1 inducer, and mRNA and protein levels for HO-1, TF and PAI-1 were examined. Total RNA was extracted from HUVEC, and was analyzed by real time RT-PCR. Protein expression levels of HO-1, TF and PAI-1 were measured by ELISA. Hemin stimulation increased HO-1 mRNA levels by 20 times. On the other hand, TF mRNA and antigen levels were minimum even after 8 hours of stimulation. Importantly, hemin stimulation reduced PAI-1 mRNA more than half after 4 hours. After HO-1 induction by hemin (100 mM) for 6 hours, HUVEC cultures were exposed to 10 ng/ml tumor necrosis factor (TNF). Prior exposure to hemin significantly increased HO-1 mRNA by 60 times in 30 minutes after stimulation with TNF. However, TNF alone could not induce HO-1 mRNA and protein levels in HUVEC. Although stimulation with TNF enhanced expressions of both TF and PAI-1 mRNA, they were significantly inhibited more than half by prior treatment with hemin. TF antigen levels were similarly decreased (5.0 to 0.7 pg/ml). PAI-1 antigen levels were also inhibited by prior treatment with hemin (1.8 to 0.1 ng/ml)(3) To see if hemin effect on HUVEC is due to HO-1 production, HO-1 inhibitor tin-protoporphyrin IX (SnPP-IX) was added to the cultures. The inhibitor effect of hemin on TF and PAI-1 productions was cancelled when HUVEC was cocultured with SnPP-IX. Conclusions - These results indicate that hemin exert inhibitory effect on TF and PAI-1 expressions through HO-1 production. Induction of HO-1 may be beneficial in the prevention of thrombosis associated with inflammation.

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Thrombin neutralizes plasminogen activator inhibitor 1 (PAI-1) that is complexed with vitronectin in the endothelial cell matrix.

The Journal of Cell Biology ◽

10.1083/jcb.115.6.1773 ◽

1991 ◽

Vol 115 (6) ◽

pp. 1773-1781 ◽

Cited By ~ 58

Author(s):

H J Ehrlich ◽

R K Gebbink ◽

K T Preissner ◽

J Keijer ◽

N L Esmon ◽

...

Keyword(s):

Endothelial Cell ◽

Plasminogen Activator ◽

Plasminogen Activator Inhibitor ◽

Cell Surface Receptor ◽

Tissue Type ◽

Plasminogen Activator Inhibitor 1 ◽

Cell Matrix ◽

Type Plasminogen Activator ◽

Activator Inhibitor ◽

Pai 1

Vitronectin endows plasminogen activator inhibitor 1 (PAI-1), the fast-acting inhibitor of both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), with additional thrombin inhibitory properties. In view of the apparent association between PAI-1 and vitronectin in the endothelial cell matrix (ECM), we analyzed the interaction between PAI-1 and thrombin in this environment. Upon incubating 125I-labeled alpha-thrombin with endothelial cell matrix (ECM), the protease formed SDS-stable complexes exclusively with PAI-1, with subsequent release of these complexes into the supernatant. Vitronectin was required as a cofactor for the association between PAI-1 and thrombin in ECM. Metabolic labeling of endothelial cell proteins, followed by incubation of ECM with t-PA, u-PA, or thrombin, indicated that all three proteases depleted PAI-1 from ECM by complex formation and proteolytic cleavage. Proteolytically inactive thrombin as well as anticoagulant thrombin, i.e., thrombin in complex with its endothelial cell surface receptor thrombomodulin, did not neutralize PAI-1, emphasizing that the procoagulant moiety of thrombin is required for a functional interaction with PAI-1. A physiological implication of our findings may be related to the mutual neutralization of both PAI-1 and thrombin, providing a new link between plasminogen activation and the coagulation system. Evidence is provided that in ECM, procoagulant thrombin may promote plasminogen activator activity by inactivating PAI-1.

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TNF-α, but not IL-6, stimulates plasminogen activator inhibitor-1 expression in human subcutaneous adipose tissue

Journal of Applied Physiology ◽

10.1152/japplphysiol.01220.2004 ◽

2005 ◽

Vol 98 (6) ◽

pp. 2019-2023 ◽

Cited By ~ 23

Author(s):

Peter Plomgaard ◽

Pernille Keller ◽

Charlotte Keller ◽

Bente Klarlund Pedersen

Keyword(s):

Adipose Tissue ◽

Mrna Expression ◽

Plasminogen Activator ◽

Subcutaneous Adipose Tissue ◽

Plasminogen Activator Inhibitor ◽

Plasminogen Activator Inhibitor 1 ◽

Tnf Α ◽

Subcutaneous Adipose ◽

Activator Inhibitor ◽

Pai 1

Plasminogen activator inhibitor-1 (PAI-1) is produced by adipose tissue, and elevated PAI-1 levels in plasma are a risk factor in the metabolic syndrome. We investigated the regulatory effects of TNF-α and IL-6 on PAI-1 gene induction in human adipose tissue. Twenty healthy men underwent a 3-h infusion of either recombinant human TNF-α ( n = 8), recombinant human IL-6 ( n = 6), or vehicle ( n = 6). Biopsies were obtained from the subcutaneous abdominal adipose tissue at preinfusion, at 1, 2, and 3 h during the infusion, and at 2 h after the infusion. The mRNA expression of PAI-1 in the adipose tissue was measured using real-time PCR. The plasma levels of TNF-α and IL-6 reached 18 and 99 pg/ml, respectively, during the infusions. During the TNF-α infusion, adipose PAI-1 mRNA expression increased 2.5-fold at 1 h, 6-fold at 2 h, 9-fold at 3 h, and declined to 2-fold 2 h after the infusion stopped but did not change during IL-6 infusion and vehicle. These data demonstrate that TNF-α rather than IL-6 stimulates an increase in PAI-1 mRNA in the subcutaneous adipose tissue, suggesting that TNF-α may be involved in the pathogenesis of related metabolic disorders.

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