Plasminogen Activator Inhibitor Type-1 Protein Research Articles

Clinical and diagnostic significance of plasminogen activator inhibitor type 1 in the early development of coronary heart disease

Aim to determine the quantitative content of the tissue plasminogen activator inhibitor type 1 (PAI-1) and to determine its relationship with risk factors for coronary artery disease.
 Material and methods. The study included 67 male and female patients (36 men and 31 women) with coronary artery disease (CAD) and stable angina classes II-IV with the presence of hypertension. The mean age of the patients was 60.20.76 years. In all patients, clinical and anamnestic data were collected during the examination. For pain assessment, we used a verbal scale. We registered the anthropometric data and body mass index. Biochemical and instrumental methods of research were used according to the standards for diagnosing CAD. We assessed the occurrence of risk factors in patients included in the study. A complex of general clinical laboratory and instrumental tests was used, as well as the method of enzyme immunoassay ELISA, which determined the level of PAI-1, platelet aggregation activity, renin, aldosterone, cortisol and endothelin.
 Results. The group of patients demonstrated a reliable elevation of PAI-1 plasma level. Compared with practically healthy people, the level of this biomarker was 1.6 times higher in patients with coronary artery disease. A significant elevation of PAI-1 level indicates an increased risk of endothelial and hemostasiological disorders in patients, which, in turn, increases the risk of thrombogenic complications. The study revealed the correlation of PAI-1 protein in CAD patients with such risk factors as mixed anxiety-depressive disorder, smoking, hypertension, obesity, and hypercholesterolemia.

Independent validation of stromal uPA in ABCSG-08: Level 1b evidence for the prognostic value of uPA immunohistochemistry

PurposeTo validate the prognostic role of urokinase-type plasminogen-activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) protein expression in FFPE archived tumor samples when assessed by immunohistochemistry. Patients and methodsFresh-frozen, paraffin-embedded (FFPE) samples from 303 postmenopausal women with hormone receptor-positive, early breast cancer were investigated. The patients had received 5 years of endocrine therapy in the prospectively randomized ABCSG-8 trial. Immunohistochemistry for stromal uPA and PAI-1 protein expression was correlated with distant recurrence-free survival (DRFS) and overall survival (OS). ResultsWe detected stromal uPA in 132 of 297 tumors (44.4%) and stromal PAI-1 expression in 74 out of 299 samples (24.7%). Co-expression of uPA and PAI-1 was present in 48 of 294 (16.3%) cases. Neither uPA nor PAI-1 expression was associated with tumor size, age, nodal status, grading, or quantitative receptor status. Patients whose tumor stroma expressed uPA protein had a significantly shorter DRFS (adjusted HR for relapse: 2.78; 95% CI 1.31–5.93; p = 0.008 Cox regression analysis) than women without uPA expression. No such association was seen for PAI-1 and the uPA/PAI1 ratio. After a median follow-up of 5.6 years, women with uPA-positive tumors demonstrated significantly shorter DRFS (93.3% vs. 84.8%; p < 0.013 log-rank test), and tended to have a worse OS (83.0% vs. 77.3%; p = 0.106) compared to women with uPA negative tumors. ConclusionThis independent validation in archived tumor samples from a large prospective randomized trial confirms the clinical utility of stromal uPA evaluation by immunohistochemistry. This provides level 1b evidence for the prognostic role of stromal uPA in women with endocrine-responsive early breast cancer.

Effects of 17β-Estradiol on the Plasminogen Activator System in Vascular Smooth Muscle Cells Treated with Lysophophatidylcholine.

ObjectivesWhen administered soon after menopause, hormone therapy can prevent coronary heart diseases in women. To explore the mechanism underlying the cardioprotective actions of estrogen, we investigated the effects of 17β-estradiol (17β-E2) on the plasminogen activator system using cultured vascular smooth muscle cells (VSMCs).MethodsVSMCs were isolated from rat aortas. Protein expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were evaluated using Western blotting and enzyme-linked immunosorbent assay, respectively. The enzyme activity of PAI-1 in a conditioned medium was assessed via reverse fibrin overlay zymography and that of t-PA was assessed via fibrin overlay zymography. Gene expression was quantified using real-time reverse transcription-polymerase chain reaction.ResultsFollowing pre-treatment for 24 hours, 17β-E2 suppressed both protein expression and enzyme activity of PAI-1 stimulated by lysophosphatidylcholine (lysoPC) in a significant and dose-dependent manner at a near physiological concentration. Moreover, 17β-E2 (10−7 M) inhibited PAI-1 gene expression, and ICI 182,780—a specific estrogen receptor antagonist—blocked the effects of 17β-E2 on the PAI-1 protein. 17β-E2 did not affect t-PA secretion but significantly enhanced free t-PA activity through reduced binding to PAI-1. Furthermore, 17β-E2 suppressed intracellular reactive oxygen species production and nuclear factor-κB-mediated transcription.ConclusionsIn VSMCs stimulated with lysoPC, 17β-E2 reduced PAI-1 expression through a non-receptor-mediated mechanism via antioxidant activity as well as a receptor-mediated mechanism; however, it did not alter t-PA secretion. Of note, 17β-E2 suppressed PAI-1 activity and concurrently enhanced t-PA activity, suggesting a beneficial influence on fibrinolysis.

MiR-30c may serve a role in endometriosis by targeting plasminogen activator inhibitor-1.

The present study aimed to investigate the role of miR-30c in endometriosis (EMs) and the underlying mechanism. The expression of miR-30c and plasminogen activator inhibitor type 1 (PAI-1) mRNA in EMs tissues was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and the expression of PAI-1 protein was detected by western blot analysis. The proliferation, migration, invasion and adhesion of endometrial stromal cells (ESCs) in different groups transfected with miR-30c mimic or inhibitor were compared. It was demonstrated that miR-30c expression in ectopic and eutopic endometriosis tissues were significantly lower than in normal endometrial tissue. However, PAI-1 mRNA expression in ectopic and eutopic endometrial tissues was higher than in normal endometrial tissues. Furthermore, the expression of PAI-1 protein was higher in ectopic and eutopic endometrosis tissues than in normal tissues. RT-qPCR results indicated that miR-30c expression was significantly increased or decreased in ESCs following transfection of mimic or inhibitor of miR-30c, respectively. Overexpression of miR-30c repressed the expression of PAI-1 mRNA and protein, while inhibition of miR-30c upregulated the expression of PAI-1 in ESCs. In addition, the invasion, migration, proliferation and adhesion of ESCs was repressed following the overexpression of miR-30c, whereas they were promoted when miR-30c expression was downregulated. The results of the present study indicated that miR-30c serves an important role in the development and progression of EMs by regulating the expression of PAI-1.

Expression, Intracellular Localization, and Prognostic Value of Plasminogen Activator Inhibitor 1 and PAI-1 RNA-Binding Protein 1 in Primary and Recurrent Ovarian Cancer: A Study of the Tumor Bank Ovarian Cancer Network

Background: The plasminogen activator system plays a key role in ovarian cancer (OC) tumor progression. The plasminogen activator inhibitor type 1 (PAI-1) and the recently identified PAI-1 RNA binding protein 1 (PAI-RBP1) are primary regulators of plasminogen activation and thus are putative biomarkers for OC progression. Methods: One hundred fifty six OC patients were analyzed to identify the presence of PAI-1 and PAI-RBP1 and subsequently correlated to clinicopathological parameters. Primary cells obtained from OC patient samples were applied in fluorescence microscopy analysis for examination of PAI-1 and PAI-RBP1 distribution. Results: PAI-1 and PAI-RBP1 have been found to be predictive markers for OC patients' outcome. PAI-1 levels significantly correlated with volume of ascites, FIGO staging, and lymph node status. PAI-RBP1 expression significantly correlated with age at first diagnosis, histological tumor type, presence of distant metastasis (pM), and recurrence. PAI-1 showed a trend toward association and PAI-RBP1 was significantly associated with progression-free survival. Notably, PAI-1 protein in recurrent OC tissues was exclusively localized in the nucleus. Conclusion: This study has shown that a combination of PAI-1 and PAI-RBP1 may represent novel prognostic factor for OC. Prospective trials are needed.

Podocyte injury-driven intracapillary plasminogen activator inhibitor type 1 accelerates podocyte loss via uPAR-mediated β1-integrin endocytosis.

Podocyte-endothelial cell cross-talk is paramount for maintaining the filtration barrier. The present study investigated the endothelial response to podocyte injury and its subsequent role in glomerulosclerosis using the podocyte-specific injury model of NEP25/LMB2 mice. NEP25/LMB2 mice showed proteinuria and local podocyte loss accompanied by thrombotic microangiopathy on day 8. Mice showed an increase of glomerular plasminogen activator inhibitor type 1 (PAI-1) mRNA and aberrant endothelial PAI-1 protein already on day 1, before thrombosis and proteinuria. A PAI-1-specific inhibitor reduced proteinuria and thrombosis and preserved podocyte numbers in NEP25/LMB2 mice by stabilization of β1-integrin translocation. Heparin loading significantly reduced thrombotic formation, whereas proteinuria and podocyte numbers were unchanged. Immortalized podocytes treated with PAI-1 and the urokinase plasminogen activator (uPA) complex caused significant cell detachment, whereas podocytes treated with PAI-1 or uPA alone or with the PAI-1/uPA complex pretreated with an anti-uPA receptor (uPAR) antibody failed to cause detachment. Confocal microscopy and cell surface biotinylation experiments showed that internalized β1-integrin was found together with uPAR in endocytotic vesicles. The administration of PAI-1 inhibitor or uPAR-blocking antibody protected cultured podocytes from cell detachment. In conclusion, PAI-1/uPA complex-mediated uPAR-dependent podocyte β1-integrin endocytosis represents a novel mechanism of glomerular injury leading to progressive podocytopenia. This aberrant cross-talk between podocytes and endothelial cells represents a feedforward injury response driving podocyte loss and progressive glomerulosclerosis.

Plasminogen activator inhibitor type 1 expression induced by lipopolysaccharide of Porphyromonas gingivalis in human gingival fibroblast

In the gingival tissues of patients with periodontitis, inflammatory responses are mediated by a wide variety of genes. In our previous screening study, plasminogen activator inhibitor type 1 (PAI-1) mRNA binding protein expression was increased in gingiva from periodontitis patients. In this study, we further investigated the signaling pathway involved in PAI-1 expression induced by Porphyromonas gingivalis LPS (Pg LPS) in human gingival fibroblasts (HGF). When HGFs were treated with Pg LPS, both PAI-1 mRNA expression and PAI-1 protein were induced in a dose-dependent manner. Pg LPS induced NF-κB activation and the expressions of PAI-1 mRNA and protein were suppressed by pretreating with a NF-κB inhibitor. Pg LPS also induced ERK, p38, and JNK activation, and Pg LPS-induced PAI-1 expression was inhibited by ERK/p38/JNK inhibitor pretreatment. In conclusion, Pg LPS induced PAI-1 expression through NF-κB and MAP kinases activation in HGF.

Effects of black cohosh on the plasminogen activator system in vascular smooth muscle cells

ObjectiveThe rhizome of the Cimicifuga racemosa plant (commonly known as black cohosh) has been used for menopausal complaints. Studies regarding the cardiovascular effects of black cohosh are lacking. We investigated the effect of black cohosh on the plasminogen activator system in cultured vascular smooth muscle cells (VSMCs). MethodsVSMCs were isolated from rat aortae. Expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) proteins were evaluated by Western blot analysis and enzyme-linked immunosorbent assay, respectively. The activities of PAI-1 and t-PA in the conditioned media were assessed by fibrin overlay zymography. A 40% 2-propanol extract of black cohosh was used. ResultsBlack cohosh extract (BcEx) stimulated the protein expression of PAI-1, but it did not affect that of t-PA. Vitamin E, a potent antioxidant, inhibited the BcEx-induced increase in PAI-1 expression, while ICI 182,780, an estrogen receptor antagonist, had no effect. Fibrin overlay zymography revealed that BcEx increased the activity of PAI-1 in the conditioned media, while concurrently decreasing that of free t-PA by inducing a binding to PAI-1. ConclusionsBcEx induces PAI-1 protein expression in the VSMCs likely via an oxidant mechanism. It also stimulates the enzyme activity of PAI-1 and reduces that of free t-PA. These findings suggest that black cohosh might exert a negative influence on fibrinolysis.

Abstract 1922: Plasminogen activator inhibitor type-1 (PAI-1) enhances bladder cancer tumor progression.

Abstract Introduction: Bladder cancer (BCa) is among the five most common malignancies worldwide and has the highest recurrence rate of any tumor type. BCa biology is poorly understood and early detection remains one of the most urgent issues in BCa research. Previously we have identified a preliminary molecular signature of BCa in voided urine samples from BCa patients. Our results revealed aberrant overexpression of the serine protease inhibitor (serpin) plasminogen activator inhibitor type-1 (PAI-1), both at the genomic and protein level. Increasing evidence indicate that elevated levels of PAI-1 correlates to a poor prognosis in many tumor types including breast, colorectal, gastric, ovarian, and more recently bladder cancer. PAI-1 has emerged as a potential target for BCa treatment and the aim of this study is to provide a greater understanding of its role in bladder cancer progression. Methods: PAI-1 expression levels in UROtsa (benign urothelial cell), KU7 (low-grade papillary urothelial cancer cell), and UMUC14 and T24 (high-grade invasive urothelial cancer cell lines) was tested by real-time PCR (RTPCR) and Western blot analysis. Secreted PAI-1 levels were measured by ELISA. KU7 stable clones overexpressing PAI-1 was established by transfection of plasmid DNA encoding human PAI-1. T24 and UMUC14 stable clones with shRNA knockdown of PAI-1 were established by infection with retroviral particles. Cell proliferation, migration, invasion, and apoptosis assays were performed in stable clones. Results: RTPCR analysis revealed an increase of PAI-1 in T24 and UMUC14 cells (27.9 and 5.2 times, respectively) while KU7 cells exhibited significantly reduced levels as compared to the UROtsa control. Similar results were observed for PAI-1 protein concentrations in cell culture supernatants and western blot analysis. While overexpression of PAI-1 in KU7 cells promoted an increase in proliferation, migration and invasion, these were significantly reduced upon inactivation of PAI-1 in T24 and UMUC14 cells stably expressing shRNA PAI-1. Loss of PAI-1 function also led to a substantial increase in apoptotic cell death. Conclusions: These results suggest that PAI-1 overexpression in BCa promotes tumor progression and therapeutic strategies targeting PAI-1 may serve clinically beneficial outcomes. Funding provided by research grants from Florida Department of Health James and Esther King Team Science Award 10KT-01 (CJR) and National Cancer Institute RO1 CA116161 (SG). Citation Format: Evan Gomes, Steven Goodison, Makito Miyake, Shanti Ross, Ge Zhang, Charles Rosser. Plasminogen activator inhibitor type-1 (PAI-1) enhances bladder cancer tumor progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1922. doi:10.1158/1538-7445.AM2013-1922

Posttranscriptional regulation of expression of plasminogen activator inhibitor type-1 by sphingosine 1-phosphate in HepG2 liver cells

Altered expression of plasminogen activator inhibitor type-1 (PAI-1), a major physiologic inhibitor of fibrinolysis, is implicated in the progression of atherosclerosis. Sphingosine 1-phosphate (S1P) regulates expression of diverse genes and alters expression of PAI-1 in several types of cells. However, the nature of posttranscriptional regulation of expression of PAI-1 by S1P has not yet been thoroughly elucidated. The present study was undertaken to determine whether S1P has important effects on the posttranscriptional regulation of PAI-1 expression. To evaluate this possibility, we determined promoter activity, mRNA levels, 3′-untranslated region (UTR) activity, and protein levels of PAI-1 in HepG2 cells. S1P increased PAI-1 promoter activity and the expression of PAI-1 mRNA within 4h of exposure. It decreased the expression of PAI-1 mRNA and the accumulation of PAI-1 protein into the media in 24h. Human PAI-1 mRNA exists in two subspecies (3.2 and 2.2kb). S1P decreased the baseline luciferase activity of the 1kb fragment of the 3′ terminus (+2177 to 3176nt) of the 3′-UTR of the 3.2kb PAI-1 mRNA [3′-UTR (+2177–3176)]. S1P decreased expression of PAI-1 protein, presumably by regulating PAI-1 expression at the posttranscriptional level thereby affecting mRNA stability. SERPINE1 mRNA binding protein (SERBP1) and ARE3 in the 3′-UTR were involved in the posttranscriptional regulation by S1P. Our data suggest that S1P can destabilize 3.2kb PAI-1 mRNA through specific effects on the 3′-UTR. These effects appear to involve SERBP1 leading to decreased expression of PAI-1 protein.

Modulators of induction of plasminogen activator inhibitor type-1 in HepG2 cells by transforming growth factor-β

An increased expression of plasminogen activator inhibitor type-1 (PAI-1) has been implicated in accelerating atherogenesis and coronary artery disease in patients with type 2 diabetes. Transforming growth factor (TGF)-β increases its expression. An increased PAI-1 appears to predispose also to augmented fibrosis potentially contributing to negative left ventricular remodeling and heart failure after myocardial infarction. Diabetes is well known to induce oxidative stress. To elucidate molecular mechanisms underlying an increased PAI-1 production, the effects of TGF-β and oxidative stress implicated as agonists of PAI-1 synthesis were characterized with the use of human liver-derived HepG2 cells. PAI-1 mRNA was assayed by real-time PCR, and PAI-1 protein was assayed by western blotting. PAI-1 promoter (-825 -+42 bp) activity was assessed with the luciferase assay. The role of the 3'-untranslated region was delineated with the use of luciferase constructs containing the 3'-untranslated region. Oxidative stress was measured after loading carboxy-2,7-dichlorodihydrofluorescein into cells. TGF-β increased oxidative stress, which was accompanied by increases in NADPH oxidase 3 mRNA and membrane translocation of Rac proteins. TGF-β-inducible increases in the PAI-1 promoter activity involved Smad-binding elements and a nuclear factor-κB-binding site. TGF-β did not increase the activity of the PAI-1 mRNA 3'-untranslated region. TGF-β-inducible PAI-1 expression was attenuated by simvastatin and curcumin, a natural polyphenol. TGF-β can increase the expression of PAI-1 through multiple mechanisms involving Smad and nuclear factor-κB pathways and oxidative stress. As both oxidative stress and PAI-1 production were reduced by simvastatin and curcumin, modulation of oxidative stress and PAI-1 production are attractive targets for pharmacotherapy of cardiovascular disorders associated with an increased PAI-1 including type 2 diabetes and its associated consequences including accelerated coronary artery disease and an increased fibrosis that may exacerbate adverse left ventricular remodeling after myocardial infarction.

Posttranscriptional regulation of expression of plasminogen activator inhibitor type-1 by cAMP in HepG2 liver cells

Altered expression of plasminogen activator inhibitor type-1 (PAI-1), a physiologic fibrinolysis inhibitor, is implicated in atherosclerosis. Cyclic adenosine monophosphate (cAMP) alters PAI-1 expression in several cells. Nevertheless, posttranscriptional regulation of PAI-1 has not been elucidated. To determine whether cAMP affects PAI-1 expression at posttranscriptional level, we determined promoter activity, mRNA levels, 3'-untranslated region (UTR) activity and protein levels of PAI-1 using HepG2 cells. cAMP decreased PAI-1 promoter activity at 24 h and mRNA expression at 4 h while it increased mRNA expression and accumulation of PAI-1 protein into media at 24 h. Human PAI-1 mRNA exists in two subspecies (3.2 and 2.2 kb), and cAMP increased baseline luciferase activity of 3'-UTR of the 3.2 kb PAI-1 mRNA [3'-UTR (+1358-3176)] and 1 kb fragment of 3'-terminus of 3'-UTR of 3.2 kb mRNA [3'-UTR (+2177-3176)]. cAMP increased PAI-1 protein expression despite decrease in promoter activity, presumably by regulating PAI-1 expression at the posttranscriptional level and thereby affecting mRNA stability. The 53-nt fragment in 3'-UTR (+2591 to +2643 nt) was involved in posttranscriptional regulation by cAMP. Thus, cAMP can stabilize 3.2 kb PAI-1 mRNA mediated by specific effects on 3'-UTR, and these effects are associated with increased expression of PAI-1 protein.

Apelin protects against angiotensin II-induced cardiovascular fibrosis and decreases plasminogen activator inhibitor type-1 production

To test the hypothesis that apelin protects against angiotensin II (Ang II)-induced cardiovascular fibrosis and vascular remodeling. Wild-type mice administered apelin or apelin along with Ang II exhibited less cardiovascular fibrosis and decreased plasminogen activator inhibitor type-1 (PAI-1) gene expression than mice receiving Ang II, N-nitro-L-arginine methyl ester (L-NAME), apelin plus L-NAME or apelin plus Ang II plus L-NAME. In-vitro analysis using a luciferase construct driven by 3.1 kb of the human PAI-1 promoter revealed that apelin blocked Ang II-mediated PAI-1 gene expression. Immunoblotting for phosphorylated myosin phosphatase subunit and myosin light chain revealed that apelin blocked Ang II activation of the Rho kinase pathway, which is associated with induction of PAI-1 gene expression by Ang II. In addition, treatment of human aortic smooth muscle cells with apelin reduced PAI-1 mRNA and protein production in the presence and absence of Ang II. Conversely, L-NAME treatment attenuated the downregulation of PAI-1 by apelin in cells. Apelin protects against cardiac fibrosis and vascular remodeling through direct regulation of PAI-1 gene expression. This protective effect is mediated through the synergistic inhibition of Ang II signaling and increased production of nitric oxide by apelin. Our data extend previous findings and provide new insight into the molecular mechanisms by which apelin elicits a cardioprotective effect.

Carbon Monoxide-Releasing Molecule-Derived CO Regulates Tissue Factor, Plasminogen Activator Inhibitor Type 1, and Thrombomodulin Production by Human Endothelial Cells.

AbstractAbstract 1146 Background and purpose:Heme oxygenase-1 (HO-1) is a cytoprotective and anti-inflammatory protein that catalyzes the conversion of heme into biliverdin, free iron, and carbon monoxide (CO). HO-1 is rapidly induced by various oxidative stresses and inflammation, thereby playing an important role in the self defence system. Recently, Yachie, et al. reported the first human case of HO-1 deficiency. This patient showed prominent signs of intravascular hemolysis, endothelial cell injury, and abnormalities in the coagulation / fibrinolysis system, suggesting the involvement of HO-1 or HO-1 products, such as CO, regulation of coagulation / fibrinolysis system. The current study examined whether tricarbonyldichlororuthenium (II) dimer (CORM-2), which liberated CO in the presence of dimethyl sulfoxide (DMSO), modulates the expression of tissue factor (TF), plasminogen activator inhibitor type 1 (PAI-1) and thrombomodulin (TM) in human umbilical vein endothelial cells (HUVECs). Methods:HUVECs were pretreated with CORM-2 at the concentration of 50 μM for 3h, washed and stimulated with tumor necrosis factor-α(TNF-α, 10 ng/ml) for additional 2 or 5h. The mRNA and protein levels of TF, PAI-1 and TM in the cultured HUVECs were determined by real-time reverse transcriptase polymerase chain reaction and Western blotting. To determine whether CORM-2 affects the MAPK signaling pathways, the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase1/2 (ERK1/2) in the HUVECs were analyzed with Western blotting. Results:After TNF-α stimulation, TF mRNA levels were approximately 6-fold and PAI-1 mRNA levels were approximately 8-fold increased, and TM mRNA levels were decreased by 20% compared to the control. Similarly to the mRNA expression, TF and PAI-1 protein levels were increased while the TM protein level was decreased. On the other hand, pretreatment of HUVECs with CORM-2 significantly decreased TF mRNA levels (approximately 80% suppression), and PAI-1 mRNA levels (approximately 90% suppression) while increased TM mRNA levels by 3-fold as compared to the TNF-α-stimulated group (p<0.05; Figure 1). Similarly, the pretreatment with CORM-2 inhibited TNF-α-induced TF and PAI-1 protein up-regulation and TM protein down-regulation. CORM-2 inhibited TNF-α-induced activation of p38MAPK and ERK1/2 by 50% compared to the control. Conclusions:These results indicate that CO liberated by CORM-2 suppresses the TNF-α-induced TF and PAI-1 up-regulation and prevents the TNF-α-induced TM down-regulation in HUVECs via MAPK signaling pathways. Thus, the modulation of endothelial function by CO may offer a novel antithrombotic option for treatment of the hypercoagulable state associated with inflammation. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Curcumin Down-Regulates Cytokine-Mediated Tissue Factor and Plasminogen Activator Type 1 Expression In Human Endothelial Cells

AbstractAbstract 3333Curcumin (diferuloyl methane), an active component of the spice turmeric, has been shown to exhibit anti-inflammatory and antioxidant activities in addition to an anticartinogenic activity in vitro and in vivo. Recent studies reported that curcumin acts as a non-stressful and non-cytotoxic inducer of the cytoprotective heme oxigenase-1 (HO-1). HO-1 is an inducible enzyme which degrades oxidative heme into biliverdin, free iron, and carbon monoxide. The first human case of HO-1 deficiency was described from Japan by Yachie, et al (J Clin Invest 1999; 103:129). This HO-1 deficient child exhibited severe persistent endothelial damage and extremely hyper-coagulable state. These findings prompted us to study roles of HO-1 in the regulation of coagulability. Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) derived from the HO-1 deficient patient was not able to produce HO-1 production at all. In comparison to control LCLs, the HO-1 deficient LCLs expressed markedly higher levels of tissue factor (TF) mRNA and protein (2-fold, p<0.05) as well as of plasminogen activator inhibitor type 1 (PAI-1) (2-fold, p<0.05), upon exposure to oxidative stress such as hemin, suggested important roles of HO-1 or HO-1 products in the regulating of coagulation and fibrinolytic system. We then hypothesized that endothelial HO-1 induced by curcumin may ameliorate hyper-coagulable state associated with inflammation or oxidative stress. The exposure of human umbilical vein endothelial cells (HUVECs) to curcumin (15 mM) for 6 hr resulted in a significant increase in the level of both HO-1 mRNA (15-fold) and protein. The treatment of HUVECs with 15 mM curcumin for 3hr before stimulation with tumor necrosis factor-α (TNF-α, 10 ng/ml) for additional 5hr markedly inhibited TNF-a-induced expression of TF (approximately 90 % suppression) and PAI-1 (approximately 70 % suppression) (Figure1). The inhibitory effect of curcumin on the TF expression was partially abrogated by tin protoporphyrin IX (SnPP) (20 mM), an inhibitor of heme oxigenase activity, indicating that the curcumin-induced suppression of TF is partly mediated by HO-1 (Figure 1). Moreover, curcumin inhibited TNF-a-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1and 2 (ERK1/2), and c-Jun N-terminal kinase (JNK), indicating the inhibition of these signal pathways may also be responsible for the suppression of TF/PAI-1 expressions in HUVECs by curcumin. In conclusion, these results indicate that curcumin exerts anti-thrombotic effects directly and indirectly through the induction of HO-1. Curcumin may serve as a novel anti-thrombotic agent for the treatment of hyper-coagulable state or thrombus formation associated with inflammation.Figure 1. Effects of curcumin on TNF-α-induced TF and PAI-1 protein (A) and mRNA (B) expression in HUVECs. The columns represent the mean ± SD (n=3) percentages of the control culture. *p<0.05 versus TNF-α alone. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Prognostic impact of plasminogen activator inhibitor type 1 expression in bladder cancer

Recent studies have demonstrated a poor prognosis for patients who have altered expression of plasminogen activator inhibitor type 1 (PAI-1) in several cancer types. The objective of the current study was to investigate the prognostic impact of PAI-1 on patients with transitional cell carcinoma (TCC) of the urinary bladder. PAI-1 expression was quantified using real-time polymerase chain reaction in 91 TCCs and in 6 normal tissue specimens. PAI-1 concentrations were analyzed by enzyme-linked immunoadsorbent assay in plasma from 104 patients and 10 controls and in urine from 244 patients and 74 controls. PAI-1 expression was evaluated immunohistochemically in paraffin-embedded tissues (94 tumor samples and 10 adjacent normal tissue samples). The results were analyzed in relation to clinical features and follow-up. Significantly higher PAI-1 levels were detected in tissue and plasma samples, but not in urine, from patients with bladder cancer compared with controls (P=.001 and P=.008, respectively). Elevated gene expression and plasma protein concentrations were independent of tumor stage and grade. Immunostaining revealed a subgroup of patients with single tumor cells that strongly expressed PAI-1. These patients' survival was significantly shorter, and their clinical presentation was correlated significantly with lymph node-positive disease. PAI-1 gene expression in tissues and plasma protein levels were elevated in patients with TCC compared with controls. PAI-1 gene or protein expression was not associated with the clinical characteristics of bladder TCC. Although the assessment of PAI-1 expression in plasma or urine may not serve as an independent predictor of prognosis in patients with TCC, the immunohistochemical detection of single PAI-1-positive cells may serve as a predictor of survival and a possible indicator of metastasis.

Increased expression of plasminogen activator inhibitor type-1 (PAI-1) in HEPG2 cells induced by insulin mediated by the 3′-untranslated region of the PAI-1 gene and its pharmacologic implications

Insulin increases, through several molecular mechanisms, expression of plasminogen activator inhibitor-1 (PAI-1), the major physiologic inhibitor of fibrinolysis. This phenomenon has been implicated as a cause of accelerated coronary artery disease and the increased incidence of acute coronary syndromes associated with type 2 diabetes. We have previously reported that physiologic and pharmacologic concentrations of insulin induce PAI-1 synthesis in human HepG2 cells and that simvastatin can attenuate its effects. This study was performed to further elucidate mechanisms responsible for the insulin-induced PAI-1 production. Concentrations of PAI-1 mRNA were determined by real-time PCR, and PAI-1 protein was assayed by western blotting. PAI-1 promoter (-829 to +36 bp) activity was assayed with the use of luciferase reporter assays. The potential role of the 3'-untranslated region (UTR) in the PAI-1 gene was assayed with the use of luciferase constructs containing the 3'-UTR. Oxidative stress was measured by loading cells with carboxy-2,7 dichlorodihydrofluorescein. Insulin increased PAI-1 promoter activity, PAI-1 mRNA, and accumulation of PAI-1 protein in the conditioned media. Insulin-inducible PAI-1 promoter activity was attenuated by simvastatin. Experiments performed with luciferase reporters containing the 3'-UTR showed that insulin increased luciferase activity through this region. Insulin also increased oxidative stress. Both insulin-inducible luciferase activity through the 3'-UTR and oxidative stress were attenuated by simvastatin. Insulin can increase PAI-1 expression through multiple mechanisms including induction mediated by the 3'-UTR of the PAI-1 gene. Accordingly, beneficial pleiotropic effects of statins on coronary artery disease may be attributable, in part, to attenuation of overexpression of PAI-1 mediated by the 3'-UTR in syndromes of insulin resistance (such as the metabolic syndrome) and type 2 diabetes.

Post-Transcriptional Regulation of Plasminogen Activator Inhibitor Type–1 Expression in Human Pleural Mesothelial Cells

The plasminogen activator inhibitor type-1 (PAI-1) effectively blocks the activities of free and receptor-bound urokinase-type plasminogen activator. Incubation of cultured human pleural mesothelial (Met5A) cells with TGF-beta increased PAI-1 protein. TGF-beta, phorbol myristate acetate, and the translation inhibitor cycloheximide induced PAI-1 mRNA and slowed its degradation, suggesting that PAI-1 mRNA could be regulated by interaction of a PAI-1 binding protein (PAI-1 mRNABp) with PAI-1 mRNA. We found that an approximately 60 kD cytoplasmic PAI-1 mRNABp is detectable in cytoplasmic extracts of MeT5A human pleural mesothelial and malignant mesothelioma cells. The PAI-1 mRNABp specifically binds to a 33-nt sequence in the 3' untranslated region of PAI-1 mRNA. Insertion of this 33-nt sequence destabilizes otherwise stable beta-globin mRNA, indicating that the binding sequence accelerates decay of endogenous PAI-1 mRNA. Competitive inhibition by overexpression of the 33-nt binding sequence in MeT5A cells reduced PAI-1 mRNA decay and increased PAI-1 protein and mRNA expression, indicating that the PAI-1 mRNABp destabilizes PAI-1 mRNA by its interaction with the endogenous 33-nt binding sequence. Incubation of Met5A cells with TGF-beta attenuated the interaction of the PAI-1 mRNABp with the 33-nt sequence. By conventional and affinity purification, we isolated the PAI-1 mRNABp and confirmed its identity as 6-phospho-d-gluconate-NADP oxidoreductase, which specifically interacts with the full-length and the 33-nt sequence of the PAI-1 mRNA 3' untranslated region. This newly recognized pathway could influence expression of PAI-1 by mesothelial or mesothelioma cells at the level of mRNA stability in the context of pleural inflammation or malignancy.

Ghrelin attenuates plasminogen activator inhibitor‐1 production induced by tumor necrosis factor‐α in HepG2 cells via NF‐κB pathway

Plasminogen activator inhibitor type 1 (PAI-1), produced partly from liver is a risk factor for macrovascular and microvascular complications of diabetes. Ghrelin, a recently described orexigenic peptide hormone, attenuates PAI-1 induced by TNF-alpha in the human hepatoma cell line (HepG2). Exposure to TNF-alpha (1 ng/ml) for 24h caused a significant increase in PAI-1 mRNA expression and protein secretion, as evaluated by RT-PCR and ELISA, but pretreatment with ghrelin (1-100 ng/ml) inhibited both basal and TNF-alpha-induced PAI-1 release in a dose and time-dependent manner in HepG2. PDTC, selective NF-kappaB inhibitor, had no additive inhibitory effects with ghrelin. The results indicate that ghrelin inhibits both basal and TNF-alpha-induced PAI-1 production via NF-kappaB pathway in HepG2 cells, and suggest that the peptide plays a therapeutic role in atherosclerosis, especially in obese patients with insulin resistance, in whom ghrelin levels were reduced.

Prostaglandin E2 Acts via Multiple Receptors to Regulate Plasminogen-Dependent Proteolysis in the Primate Periovulatory Follicle

The ovulatory gonadotropin surge regulates expression of plasminogen activator (PA) family members within the ovarian follicle, which are implicated in follicle wall degradation at ovulation. Gonadotropin also stimulates follicular prostaglandin E2 (PGE2) production, which is required for follicle rupture. To determine whether the ovulatory gonadotropin surge regulates PA-mediated proteolysis via PGE2 in the primate follicle, monkeys received gonadotropins to stimulate follicle development. Follicular aspirates or whole ovaries were obtained before (0 h) and after human chorionic gonadotropin (hCG) administration to span the periovulatory interval. Granulosa cell levels of tissue-type PA (tPA) and PA inhibitor type 1 (PAI-1) proteins were low at 0 h hCG and higher after hCG administration. In situ zymography showed no ovarian tPA activity 0 h after hCG; tPA activity was present in granulosa cells obtained after hCG treatment. Importantly, tPA and PAI-1 proteins and tPA activity were low/nondetectable in granulosa cells obtained after treatment with hCG and the PG synthesis inhibitor celecoxib. To determine whether hCG stimulation of tPA and PAI-1 requires PGE2, granulosa cells obtained at 0 h were cultured with hCG plus indomethacin to inhibit PG production; some cells also received PGE2 or an agonist selective for one PGE2 receptor (EP). PGE2, an EP2 agonist, and an EP3 agonist increased tPA protein, whereas PGE2, an EP1 agonist, and an EP3 agonist increased PAI-1 protein. Therefore, gonadotropin increases granulosa cell tPA and PAI-1 protein levels and tPA-dependent proteolytic activity. PGE2 also increases tPA and PAI-1 protein levels in granulosa cells, suggesting that elevated PGE2 late in the periovulatory interval acts to stimulate proteolysis and follicle rupture.