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CELLULAR AND MOLECULAR
and Troglitazone Regulate Plasminogen Activator Inhibitor Type 1 Production through Extracellular Signal-Regulated Kinase- and Nuclear Factor-
B-Dependent Pathways in Cultured Human Umbilical Vein Endothelial Cells
Department of Endocrinology and Metabolism, Kanazawa University Graduate School of Medical Science, Ishikawa, Japan
Received May 12, 2003; accepted August 14, 2003.
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Abstract |
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, which induces insulin resistance. In diabetic patients, troglitazone, a thiazolidinedione, can lower the concentration of PAI-1. We investigated the TNF--induced signaling pathway that leads to PAI-1 synthesis and the target step of troglitazone in this pathway. TNF- induced PAI-1 mRNA expression and protein production in human umbilical vein endothelial cells (HUVECs). A specific inhibitor for p38 mitogen-activated protein kinase, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB 203580), and a protein kinase C inhibitor, calphostin C, had no inhibitory effects on TNF--induced PAI-1 secretion. A protein tyrosine kinase inhibitor, genistein, completely inhibited TNF--induced PAI-1 secretion, whereas an inhibitor of extracellular signal-regulated kinase (ERK) kinase, 2'-amino-3'-methoxyflavone (PD98059), and a nuclear factor-
B (NF-B) inhibitor, emodin, partly inhibited TNF--induced PAI-1 secretion. Together, PD98059 and emodin completely inhibited TNF--induced PAI-1 secretion, suggesting that both NF-B-dependent and NF-B-independent pathways are involved in TNF--induced signal pathway to PAI-1 production and that the latter pathway is mediated by activation of ERK. Furthermore, we have shown that troglitazone inhibited both TNF--induced PAI-1 protein secretion and mRNA in HUVECs. Genistein, but neither PD98059 nor emodin, was additive to the inhibitory effect of troglitazone on TNF--induced PAI-1 secretion. These results indicate That ERK and NF-B are possible targets of TNF- and troglitazone in the regulation of PAI-1 production.
) and degeneration by oxidative stress (Giugliano et al., 1996). Endothelial cells regulate blood coagulation by regulating the expression of antithrombotic factors such as glycosaminoglycans, thrombomodulin, prostacyclin, and tissue-type plasminogen activator (tPA), and expression of thrombogenic factors such as tissue factor and plasminogen activator inhibitor type 1 (PAI-1) (Engelberg, 1989). However, these endothelial regulations of coagulation disappear during atherogenesis. Plasma levels of PAI-1 are elevated in patients with thrombotic disease, coronary heart disease (Gray et al., 1993), type 2 diabetes, and hyperinsulinemia (Juhan-Vague et al., 1991), and PAI-1 expression is enhanced in atherosclerotic human arteries (Hamdan et al., 1996). This suggests that systemically and locally produced PAI-1 plays a role in the development of atherosclerosis.
PAI-1 synthesis is regulated by multiple factors. Hyperglycemia and various cytokines released from monocytes or macrophages, such as tumor necrosis factor (TNF)-, interleukin (IL)-1
, and transforming growth factor-, stimulate the release of PAI-1 from endothelial cells, thereby leading to the acceleration of coagulability (Klagsbrun and Edelman, 1989). TNF- is secreted from not only monocytes or macrophages in injured vessel walls but also from adipose tissue (Hotamisligil et al., 1993). Therefore, TNF- can greatly contribute to increased PAI-1 concentration in obese patients.
Troglitazone, a thiazolidinedione, improves glucose intolerance, insulin resistance, and dyslipidemia (Mimura et al., 1994), and so is thought to inhibit indirectly the initiation and progression of atherosclerosis. Several studies have demonstrated that troglitazone can inhibit vascular smooth muscle cell proliferation and migration (Low et al., 1996; Yasunari et al., 1997). We previously found that troglitazone directly inhibits cytokine-induced monocyte chemoattractant protein-1 (MCP-1) expression in human mesangial cells (Yokoyama et al., 2000) and endothelial cells (Yokoyama et al., 2000). It has also been reported that troglitazone improved fibrinolysis and prevented atherosclerosis by reducing PAI-1 activity in patients with diabetes mellitus (Fonseca et al., 1998) or polycystic ovary syndrome (Ehrmann et al., 1997). However, the signal transduction leading to PAI-1 production is not fully understood. In this study, we investigated TNF--induced signal transduction pathway that enhances PAI-1 synthesis and the target of troglitazone in human umbilical vein endothelial cells (HUVECs).
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Materials and Methods |
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(Pierce Endogen, Rockford, IL) and agent, control medium was removed and replaced with medium containing 0.4% FBS and antibiotics without bovine brain extract, human epidermal growth factor, and hydrocortisone. HUVECs were cultured in the medium containing 0.4% FBS with or without any of the agents in the presence of TNF- for 24 h. The cell culture supernatant of each well was collected at the time indicated and stored at –20°C until assay.
Interleukin-1 was obtained from R&D Systems (Minneapolis, MN), and phorbol ester phorbol 12-myristate 13-acetate (PMA) was from Sigma-Aldrich (St. Louis, MO). A thiazolidinedione compound, troglitazone, was from Sankyo Co., Ltd. (Tokyo, Japan). PD98059, emodin, SB 203580, calphostin C, and genistein were purchased from Calbiochem (La Jolla, CA). Their inhibition of PAI-1 secretion in the presence of troglitazone (10 µM) was assessed.
Assessment of Cell Viability. Cell viability was assessed by the trypan blue exclusion method as described previously (Yokoyama et al., 2000a,b), and by LDH release assay (CytoTox 96 nonradioactive cytotoxicity assay; Promega, Madison, MI).
To examine the reversible effect of troglitazone withdrawal on TNF--induced PAI-1 secretion, HUVECs were pretreated with or without 10 µM troglitazone in the presence of 100 ng/ml TNF- for 24 h. Thereafter, HUVECs were washed with phosphate-buffered saline three times, and changed to the medium containing 100 ng/ml TNF- without troglitazone every 24 h, and the supernatant was collected for PAI-1 measurement by ELISA until 72 h after troglitazone removal.
Antigen Assays for PAI-1 and tPA. The level of PAI-1 and tPA antigens in HUVECs culture supernatant were determined by enzyme-linked immunosorbent assay methods (TintElize PAI-1 and TintElize tPA; Biopool Inc., Umeå, Sweden) as reported previously (Declerck et al., 1988). The detection limit of these assays was 0.5 ng/ml, and the within- and between-assay coefficients of variation were 1.9 and 2.4% in a PAI-1 assay, and 5.5 and 3.5% in a tPA assay, respectively.
RNA Isolation and Northern Blot Analysis. Confluent HUVECs in culture were incubated with the medium containing 0.4% FBS with or without troglitazone (10 µM) in the presence of TNF- (100 ng/ml) for 8 h. Total RNA was prepared by a guanidium isothiocyanate-cesium chloride method (Chomczynski and Sacchi 1987), and the RNA samples (20 µg) were electrophoresed on 1.0% agarose gel containing 1.1 M formaldehyde. RNA was transferred from gel onto nylon filter (Hybond N+ filter; Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK) using 20x SSC. The probe used in this study was a complementary oligonucleotide (24-mer, 5'-GCACCAGCCGTGTCAGCTGGTCCA-3') in the coding region of the human PAI-1 mRNA sequence (Pannekoek et al., 1986). The probes were labeled by an end-labeling method with [
-32P]ATP (6,000 Ci/mmol; 1 mCi = 37 MBq) (Amersham Biosciences UK, Ltd.) and a DNA 5'-end labeling kit (MEGALABEL; Takara, Kyoto, Japan). The filter was hybridized with a 32P-labeled PAI-1 cDNA probe for 48 h at 37°C. After hybridization, the filter was washed with buffer comprising 5x SSC/0.1% SDS for 5 min at 28°C and then with buffer comprising 5x SSC/0.1% SDS for 10 min at 37°C, and finally with buffer comprising 2x SSC/0.1% SDS for 5 min at room temperature. The filter was exposed overnight to imaging analyzer film (Fuji Film, Tokyo, Japan). Hybridization signals were detected by scanning the film with a Bio imaging analyser (Fujix BAS1000; Fuji Film).
Real-Time Quantitative PCR. Complementary DNA was synthesized from total RNA as described previously (Takamura et al., 2001) and used as a template. Real-time quantitative PCR was performed by using the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA) as described previously (Ota et al., 2003). The set of primers and a TaqMan probe for PAI-1 were proprietary to Applied Biosystems (Assays-on-Demand gene expression product). To control for variation in the amount of DNA available for PCR in the different samples, PAI-1 gene expression was normalized in relation to the expression of an endogenous control, 18S ribosomal RNA (18S rRNA TaqMan control reagent kit; Applied Biosystems). The PCR conditions were 1 cycle of 50°C for 2 min, 95°C for 10 min, followed by 50 cycles of 95°C for 15 s and 60°C for 1 min.
Statistical Analysis. Data were presented as means ± S.E.M. Significance of differences between mean values was assessed by the unpaired Student's t test. P < 0.05 was considered statistically significant. All calculations were performed with the computer program, StatView, version 4.0, for Macintosh (Abacus Concepts, Berkeley, CA).
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Results |
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on PAI-1 Secretion from HUVECs. As shown in Fig. 1A, HUVECs secreted PAI-1 in the presence of 0.4% FBS in the culture medium. After the incubation with 100 ng/ml TNF-, elevations of PAI-1 levels were observed in a time-dependent manner during 24-h incubation. TNF- significantly increased the PAI-1 secretion to 1.99 ± 0.16-fold of the control value (P < 0.005 versus control), 24 h after the start of incubation. The effect of TNF- on PAI-1 secretion was dose-dependent from 1 to 100 ng/ml (Fig. 1B).
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Effect of Troglitazone on TNF--Induced PAI-1 Secretion and mRNA Expression. To examine the direct effects of troglitazone on TNF--induced PAI-1 secretion, HUVECs were cultured in the medium with or without troglitazone (1 or 10 µM) in the presence of TNF- (1, 10, and 100 ng/ml) for 24 h. As shown in Fig. 1B, troglitazone dose dependently inhibited TNF--induced PAI-1 secretion from HUVECs. Troglitazone also inhibited basal level of PAI-1 secretion (at 10 µM troglitazone, 143.0 ± 19 versus 399.3 ± 7.13 ng/ml).
To investigate whether troglitazone inhibits PAI-1 production at the level of mRNA expression in HUVECs, we performed a Northern blot analysis with RNA isolated from HUVECs cultured in the presence of TNF- with or without 10 µM troglitazone. As shown in Fig. 2, TNF- induced a marked increase in the two forms of PAI-1 mRNAs at 3.2 and 2.3 kb produced by alternative polyadenylation (Schleef et al., 1988). Troglitazone inhibited expression of both forms of TNF--induced PAI-1 mRNA.
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We also confirmed the PAI-1 mRNA expression quantitatively by using a real-time PCR method. As shown in Table 1, TNF- induced PAI-1 mRNA expression dose dependently. Troglitazone inhibited both basal and TNF--induced PAI-1 mRNA expression dose dependently.
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Meanwhile, the endothelial cell viability assessed by the trypan blue exclusion method did not differ between control and troglitazone-treated cells (control, 84.6 ± 1.5%; 1 µM troglitazone-treated group, 87.1 ± 0.9%; 10 µM troglitazone-treated group, 90.1 ± 1.9%), indicating that the doses of troglitazone used in this study were not cytotoxic to HUVECs.
To examine the reversibility of PAI-1 secretion, we evaluated PAI-1 secretions from HUVECs in the presence of TNF- every 24 h after removal of troglitazone. As shown in Fig. 3, the inhibitory effect of troglitazone on TNF--induced PAI-1 secretion lasted for 24 h after troglitazone removal, but PAI-1 secretion was gradually recovered and there was no significant difference between groups pretreated with and without troglitazone at time point 48 h. TNF--induced PAI-1 secretion was completely recovered 72 h after troglitazone removal. From the result of the cell viability and the reversibility of PAI-1 production, it becomes apparent that the inhibition of PAI-1 production was not due to a cytotoxic effect of troglitazone on HUVECs.
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Effect of TNF- and Troglitazone on tPA Secretion. Because the balance between PAI-1 and tPA determines fibrinolytic activity (Schleef RR et al., 1988), we next investigated the effects of TNF- and troglitazone on tPA production from HUVECs. TNF- essentially did not affect tPA production at any time (Fig. 4A) and any dose (Fig. 4B). Only high dose (10 µM), but not low dose (1 µM) troglitazone, significantly inhibited tPA production from HUVECs (Fig. 4B).
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Effects of Signal Transduction Inhibitors on TNF--Induced PAI-1 Secretion. To determine TNF--induced signal transduction pathway leading to PAI-1 production, we examined the effects of a variety of inhibitors on TNF--induced PAI-1 secretion with ELISA method (Fig. 5). We used the signal transduction inhibitors at concentrations that were found to be most effective and not cytotoxic in our pilot study (data not shown). A specific inhibitor for p38 mitogen-activated protein (MAP) kinase, SB 203580, and a protein kinase C (PKC) inhibitor, calphostin C, had no inhibitory effects on TNF--induced PAI-1 secretion even at the maximal doses reported previously (Laird et al., 1998; Kim et al., 2000). MAP kinase/extracellular signal-regulated kinase (ERK) kinase-specific inhibitor PD98059 treatment lowered TNF--induced PAI-1 to 80.6 ± 0.6% (P < 0.05). An NF-B inhibitor, emodin, lowered PAI-1 secretion to 44.2 ± 0.2% (P < 0.01). A protein tyrosine kinase inhibitor, genistein, completely lowered it to control level (P < 0.005). Emodin, but not genistein, had an additive inhibitory effect on TNF--induced PAI-1 with PD98059.
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Effects of Signal Transduction Inhibitors on TNF--Induced PAI-1 Secretion in the Presence of Troglitazone. To clarify the target of troglitazone in the signal transduction pathway leading to the TNF--induced PAI-1 production, in the presence of 10 µM troglitazone, we examined whether genistein, PD98059, and emodin, have additive effects on TNF--induced PAI-1 secretion with troglitazone. As shown in Fig. 6A, neither PD98059 nor emodin had additional inhibitory effects with troglitazone alone. On the other hand, genistein had an additional inhibitory effect with troglitazone (P < 0.05 versus troglitazone alone). Because genistein alone completely reduced TNF--induced PAI-1 production to the basal level (Fig. 5), we confirmed the additive effect of genistein on troglitazone. As shown in Fig. 6B, the inhibitory effects of genistein both at low and high doses were essentially additive with troglitazone.
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To confirm the specificity of the inhibitors, we checked the cell viability for all combinations of inhibitors by LDH release assay. As shown in Fig. 7, TNF- and troglitazone did not increase LDH release from HUVECs. Combinations of signal inhibitors (genistein, PD98059, and emodin), even at their maximal doses, rather were protective against LDH release from HUVECs. These results were also confirmed by trypan blue exclusion assay (data not shown). Thus, dose determination of the inhibitors seems optimal. Each inhibitor at doses used in this study reduced TNF--induced PAI-1 production by inhibiting specific signal transduction pathway, not by nonspecific cytotoxic actions.
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Inhibitory Effect of Troglitazone on IL-1- and PMA-Induced PAI-1 Secretion. To examine specificity of the inhibitory effects of troglitazone further, effects of troglitazone on PAI-1 production induced with secretagogues other than TNF- were examined. As shown in Fig. 8, IL-1 (10 ng/ml) and PMA (100 ng/ml) significantly enhanced PAI-1 secretion from HUVECs. Troglitazone (10 µM) significantly inhibited both IL-1- and PMA-induced PAI-1 secretion from HUVECs (Fig. 8).
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Discussion |
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and IL-1, released from macrophages are thought to play an important role in atherogenesis (Munro and Cortan, 1988). Because adipose tissue is a major site of synthesis of TNF- in obese humans (Hotamisligil et al., 1995), TNF- may stimulate PAI-1 production from local lesion such as vascular cells in patients with obesity and insulin resistance. We showed that TNF- increased PAI-1 mRNA expression and protein production in endothelial cells as reported previously (van Hinsbergh et al., 1988). Human tissues express both 3.2- and 2.3-kb forms of PAI-1 mRNA, due to alternative polyadenylation (Schleef et al., 1988). Cytokines such as TNF- and IL-1 increases both forms of PAI-1 mRNA, but with a preferential increase in the larger mRNA form (Schleef et al., 1988). Thus, the cytokine-induced 3.2-kb mRNA could make a larger contribution to the development of atherosclerosis.
In the intracellular signal transduction pathways inducing PAI-1 production, it is as yet unclear what happens after TNF- binds to its receptors. In the present study, PD98059 and emodin together completely inhibited TNF--induced PAI-1 secretion, whereas each alone only partly inhibited it. These findings suggest that both NF-B-dependent and -independent pathways are involved in TNF--induced PAI-1 production and that the NF-B-independent pathway is predominantly mediated by activation of ERK. Whether other transcription factors, activator protein-1, and signal transducers and activators of transcription are involved in the NF-B-independent pathway has yet to be elucidated. A protein tyrosine kinase inhibitor, genistein, completely inhibited TNF--induced PAI-1 production. van Hinsbergh et al. (1994) suggested that genistein does not inhibit receptor-coupled tyrosine kinase, because other tyrosine kinase inhibitors, tyrphostin A47 and compound 5, which inhibit receptor-linked tyrosine kinase activity, do not inhibit PAI-1 synthesis. Thus, genistein may inhibit some down-stream pathways of TNF- receptor activation to transcription of PAI-1 gene. In addition, we found that the inhibitory effect of genistein on PAI-1 secretion was not additive with PD98059, suggesting that tyrosine kinase may regulate the ERK pathway.
High levels of glucose in diabetics increase diacylglycerol levels and PKC activity in aortic endothelial cells (Inoguchi et al., 1992). We showed in this study that PKC-activating phorbol ester PMA also stimulates PAI-1 production in HUVECs as reported previously (Feener et al., 1995). PMA induces PAI-1 expression via activation of PKC and MAP kinase kinase, leading to c-Jun homodimer binding to the responsive region necessary for PMA responsiveness of PAI-1 gene promoter (Arts et al., 1999). Cytokines such as IL-1 does not stimulate this pathway, but stimulates protein tyrosine kinase in HepG2 cells. Although it was reported that TNF- signal transduction in endothelial cells involves PKC (Magnuson et al., 1989), we found no inhibitory effect by the PKC inhibitor calphostin C on TNF--induced PAI-1 secretion. This finding supports one previous report, in which TNF--induced PAI-1 secretion from endothelial cells was not inhibited by the PKC inhibitors H-7 and its structural analog HA-1004 (Niedbala and Stein-Picarella, 1993). Thus, we conclude that TNF--induced signal transduction pathway leading to PAI-1 production does not involve PKC activation.
We have shown that a thiazolidinedione agent, troglitazone, inhibits both TNF--induced PAI-1 mRNA increase and protein secretion. Troglitazone also inhibited IL-1-induced PAI-1 production. The stimulatory effect of IL-1 on PAI-1 production from HUVECs was reported not to be additive with TNF- (Schleef et al., 1988), suggesting that targets of troglitazone involve a common cytokine signal transduction pathway for PAI-1 production.
Besides our observations, there are some reports suggesting that thiazolidinediones have the potential to act on vascular cells directly: 1) troglitazone inhibits high glucose-induced proliferation and migration of coronary smooth muscle cells (Yasunari et al., 1997); 2) troglitazone inhibits intimal hyperplasia in a balloon-injury model of rat aorta (Low et al., 1996); and 3) troglitazone and pioglitazone inhibit endothelial cell proliferation (Granlinski et al., 1998). In addition, we previously showed that troglitazone inhibits cytokine-induced MCP-1 production in human mesangial cells (Yokoyama et al., 2000a) and endothelial cells (Yokoyama et al., 2000b), suggesting that it might be useful for inhibition of glomerulosclerosis and atherosclerosis. These reports suggest that a thiazolidinedione not only indirectly inhibits atherosclerosis by improving glucose intolerance, insulin resistance, and dyslipidemia but also has direct effects on endothelial cells. The fact that plasma concentration of PAI-1 is reduced after troglitazone is administered into type 2 diabetic patients (Fonseca et al., 1998) might be explained partly by the direct effects of troglitazone on endothelial cells. Therefore, a thiazolidinedione may be good for the preventing macro- and microangiopathy in diabetes.
The report by Kato et al. (1999) suggests that thiazolidinediones inhibit TNF--induced PAI-1 mRNA expression, possibly through a peroxisome proliferator-activated receptor (PPAR)--mediated mechanism. However, one report describes that PPAR- activation in endothelial cells increases PAI-1 expression (Marx et al., 1999). Thus, the target signal transduction pathway of thiazolidinedione in the regulation of PAI-1 production is still remains unclear, although one report suggested that troglitazone inhibits MAP kinase activity in human aortic smooth muscle cells (Kihara et al., 1998). In the present study, we found that genistein, but neither PD98059 nor emodin, was additive to the inhibitory effect of troglitazone on TNF--induced PAI-1 secretion. These findings suggest that troglitazone could inhibit TNF--induced PAI-1 production via protein tyrosine kinase-independent pathway and that troglitazone at least partly inhibits TNF--induced activation of ERK and NF-B. A thiazolidinedione is a direct ligand for PPAR, a member of the nuclear receptor superfamily of ligand-dependent transcription factors (Tontonoz et al., 1994). PPAR- inhibits gene expression in part by antagonizing the activities of transcription factors activator protein-1, signal transducers and activators of transcription-1, and NF-B (Ricote et al., 1998). Troglitazone also inhibits TNF--induced MCP-1 production (Yokoyama et al., 2000a,b), which is known to be mediated by NF-B (Rovin et al., 1995). Chen and Han (2001) reported troglitazone inhibits TNF--induced ICAM-1 gene expression by suppressing NK-B/DNA binding (Chen et al., 2001). Recently, it was reported that intranuclear and total cellular NF-B content fell in mononuclear cells of obese patients in whom plasma PAI-1 levels decreased after troglitazone therapy (Ghanim et al., 2001).
High glucose-induced PKC activation plays a role in the development of diabetic vascular complications (Inoguchi et al., 1992). We showed that troglitazone significantly inhibits PMA-induced PAI-1 secretion from HUVECs, suggesting that troglitazone could also target PKC/MAP kinase pathway and could contribute to improve a high glucose-induced atherogenic property. Our data shown in Fig. 5 suggested that the ERK pathway does not mainly contribute to TNF--induced PAI-1 secretion. Other members of MAP kinase family, probably c-Jun NH2-terminal kinase (JNK) may be involved in the TNF--induced signal transduction pathway. Recently, JNK has been shown to be phosphorylated by PPAR- in vitro (Camp et al., 1999). As TNF- activates JNK (Modur et al., 1996), it is a potential pathway on which troglitazone may act.
Because the balance between PAI-1 and tPA determines hypofibrinolysis and procoagulant activity (Schleef et al., 1988), we investigated the effects of TNF- and troglitazone on tPA production from HUVECs. TNF- essentially did not affect tPA production. Considering that TNF- strongly stimulates PAI-1 production, TNF- seems to inhibit the net fibrinolytic system in HUVECs. In this regard, however, it is still controversial whether TNF- inhibits or even stimulates the tPA production from endothelial cells. Schleef et al. (1988) reported an inhibitory effect of TNF- on tPA antigen production from HUVECs. On the other hand, Kawai et al. (1996) demonstrated that TNF- rather stimulates tPA production in the presence of shear stress in HUVECs. In our preliminary study, responses of tPA production from HUVECs differ between different lots of HUVECs (data not shown). Thus, effects of TNF- on tPA production and net fibrinolytic system should be carefully examined further. In this study, only high dose (10 µM), but not low dose (1 µM) troglitazone significantly inhibited tPA production from HUVECs. Considering the ratios of PAI-1 to tPA, troglitazone at 1 µM seems to have a beneficial effect on TNF--induced alterations in fibrinolytic system. However, further comprehensive study also should be required to elucidate whether troglitazone has beneficial or harmful effects on fibrinolytic systems both in vitro and in vivo.
In conclusion, we have demonstrated that ERK and NF-B are involved in TNF--induced signaling pathway leading to PAI-1 production. Troglitazone inhibited TNF--induced PAI-1 mRNA expression and protein secretion possibly by inhibiting, at least in part, both ERK- and NF-B-dependent pathways. In addition, protein tyrosine kinase could be a potential therapeutic target to control PAI-1 production. These findings will help in the development of novel pharmacological interventions for PAI-1 regulation and atherosclerosis.
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Footnotes |
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B, nuclear factor-B; LDH, lactate dehydrogenase; ELISA, enzyme-linked immunosorbent assay; SSC, standard saline citrate; PCR, polymerase chain reaction; kb, kilobase; MAP, mitogen-activated protein; PKC, protein kinase C; ERK, extracellular signal-regulated kinase; PPAR-, peroxisome proliferator-activated receptor-; JNK, c-Jun N-terminal kinase. Address correspondence to: Dr. Toshinari Takamura, Department of Endocrinology and Metabolism, Kanazawa University Graduate School of Medical Science, 13-1 Takara-machi, Kanazawa, Ishikawa 920-8641, Japan. E-mail: tt{at}medf.m.kanazawa-u.ac.jp
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