Plasminogen Activator Inhibitor Type-1 Expression Research Articles

P421 The faecal biomarker LDN-051 is a novel tool for monitoring disease activity and therapeutic response in Inflammatory Bowel Diseases

Abstract Background Crohn’s disease and ulcerative colitis, two forms of inflammatory bowel disease (IBD) require lifelong treatment and patient monitoring. Current predictors of relapse and therapeutic success have limitations, therefore there is high unmet need to identify novel biomarkers in IBD. Monitoring approaches for IBD are mostly invasive, time-consuming, and expensive. Therefore, a simple, rapid, and non-invasive test, with ability to differentiate IBD from other gastrointestinal conditions and to monitor disease activity, will have important clinical implications. Our previous mucosal cytokine profiling identified that Plasminogen activator inhibitor type 1 (PAI-1) is expressed in IBD patients with active disease, but not in control. Therefore, our aim was to assess the utility of PAI-1 in monitoring disease activity and therapeutic response of IBD patients in different biological samples. Methods Serum, biopsy and faecal samples were collected from 132 IBD patients and 30 controls without IBD. ELISA method was used to define the level of PAI-1 in the mucosa, serum, and faecal samples and the serum CRP levels were also determined. For the mucosal gene expression analysis qRT-PCR was used. The correlation and ROC analyses were applied to observe the relationship between the disease activity and PAI-1 level. Results The applied screening approach detected significant higher expression of PAI-1 in active IBD. We demonstrated that the serum, mucosal and faecal PAI-1 concentration was significantly elevated in IBD patients showing clinical and endoscopic activity. Additionally, in responder patients the initial PAI-1 level decreased significantly upon successful therapy, whereas it remained unchanged in non-responders. We also showed that faecal PAI-1 selectively increases in active IBD patients, but not in other organic gastrointestinal diseases. In addition, the stability analysis of faecal PAI-1 showed that the protein was detectable up to one week at room temperature and 4ºC. The serum, mucosal and faecal PAI-1 level showed positive correlation to the endoscopic activity. ROC curves demonstrated that faecal PAI-1 showed a relatively high power to distinguish between active and control patients. However, the serum CRP level no reflected to the therapeutic response. Conclusion Our results suggest that serum, mucosal and faecal PAI-1 expression reflects to the disease activity in IBD, which could be used to disease monitoring and possibly therapeutic response. The correlation between the faecal PAI-1 level and the endoscopic activity promotes that it could be used as a novel non-invasive disease specific faecal biomarker in the IBD diagnosis.

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.

Plasminogen activator inhibitor type-1 is a negative regulator of hematopoietic regeneration in the adipocyte-rich bone marrow microenvironment

Bone marrow adipocytes (BMAs) have recently been recognized as a niche component with a suppressive function. Obese individuals with abundant BMAs exhibit impaired hematopoietic regeneration after hematopoietic stem cell transplantation (HSCT). We hypothesized that plasminogen activator inhibitor type-1 (PAI-1), an adipokine that regulates the fibrinolytic system, contributes to impaired hematopoiesis in bone marrow (BM) microenvironment with abundant BMAs. We demonstrated that BMAs differentiated in vitro could secrete PAI-1 and were positive for PAI-1 in vivo. In addition, the abundance of BMAs was associated with high levels of PAI-1 expression. The BMA-rich microenvironment exhibited impaired hematopoietic regeneration after HSCT when compared with a BMA-less microenvironment. The impaired hematopoietic regeneration in BMA-rich microenvironment was significantly alleviated by PAI-1 knockout or PAI-1 inhibitor treatment. Obese mice with abundant BMAs, compared with normal-weight mice, exhibited higher bone marrow PAI-1 concentrations, increased fibrinolytic system suppression, and lower stem cell factor (SCF) concentrations after HSCT. PAI-1 inhibitor administration significantly activated the fibrinolytic system in obese mice, contributing to the higher SCF concentration. Moreover, PAI-1 inhibitor treatment significantly alleviated the impaired hematopoietic regeneration in obese mice both after 5-fluorouracil injection and HSCT. These results indicate that PAI-1 hinders hematopoietic regeneration in BMA-rich microenvironments. The blockade of PAI-1 activity could be a novel therapeutic means of facilitating hematopoietic reconstitution in BMA-rich patients.

CLINICOPATHOLOGICAL AND PROGNOSTIC SIGNIFICANCE OF PAI-1 EXPRESSION IN ORAL TONGUE SQUAMOUS CELL CARCINOMA

Objective: The purpose of this study was to evaluate the expression of plasminogen activator inhibitor type-1 (PAI-1) and its association with clinicopathological parameters and survival rates of oral tongue squamous cell carcinoma (OTSCC). Study Design: Sixty cases of OTSCC were selected for morphologic and immunohistochemical study. Each case was histopathologically graded according to Almangush et al. (2015). Cytoplasmic and membrane immunoexpression of PAI-1 were semiquantitatively analyzed at the deep invasive front. Pearson χ2 and Fisher exact test were performed to investigate the relationship between immunohistochemical findings and clinicopathological characteristics. Survival analyses were performed using the Kaplan-Meier method and log-rank test. Results: PAI-1 was highly expressed by neoplastic and stromal cells. No statistically significant associations were found between cytoplasmic immunopositivity for PAI-1 and clinical stage, nodal metastasis, local recurrence, disease outcome, or histopathological grade of malignancy (P> .05). Membrane immunopositivity for PAI-1 was significantly associated with tumor budding and histopathological risk-assessment score (P< .05). Cytoplasmic and membrane expression of PAI-1 were not associated with disease-specific or disease-free survival (P> .05). Conclusion: Our findings suggest that PAI-1 partly influences the biological behavior of OTSCC. However, the immunoexpression of this protein may not be useful to predict survival of the studied lesion. Objective: The purpose of this study was to evaluate the expression of plasminogen activator inhibitor type-1 (PAI-1) and its association with clinicopathological parameters and survival rates of oral tongue squamous cell carcinoma (OTSCC). Study Design: Sixty cases of OTSCC were selected for morphologic and immunohistochemical study. Each case was histopathologically graded according to Almangush et al. (2015). Cytoplasmic and membrane immunoexpression of PAI-1 were semiquantitatively analyzed at the deep invasive front. Pearson χ2 and Fisher exact test were performed to investigate the relationship between immunohistochemical findings and clinicopathological characteristics. Survival analyses were performed using the Kaplan-Meier method and log-rank test. Results: PAI-1 was highly expressed by neoplastic and stromal cells. No statistically significant associations were found between cytoplasmic immunopositivity for PAI-1 and clinical stage, nodal metastasis, local recurrence, disease outcome, or histopathological grade of malignancy (P> .05). Membrane immunopositivity for PAI-1 was significantly associated with tumor budding and histopathological risk-assessment score (P< .05). Cytoplasmic and membrane expression of PAI-1 were not associated with disease-specific or disease-free survival (P> .05). Conclusion: Our findings suggest that PAI-1 partly influences the biological behavior of OTSCC. However, the immunoexpression of this protein may not be useful to predict survival of the studied lesion.

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.

Bone Marrow Adipocyte-Derived PAI-1 As a Negative Regulator of Hematopoietic Regeneration

Introduction: Bone marrow adipocytes (BMAs) reportedly act as negative regulators of hematopoietic regeneration. This can be observed in obese or elderly patients, both of whom exhibit BMA-rich microenvironment with poor hematopoietic regeneration. Though this relationship is known, the mechanism(s) underlying the suppressive effects of BMAs on hematopoietic regulation are yet to be elucidated. Recently, in our previous study, we demonstrated that blocking plasminogen activator inhibitor type-1 (PAI-1) enhances hematopoietic regeneration through the activation of the fibrinolytic pathways in the bone marrow. As PAI-1 is known as one of the major adipokines, we reasoned that PAI-1 produced from BMAs could have a causative role in the impairment of hematopoietic regeneration in the BMA-rich microenvironment. Methods: C57B6/J and PAI-1-deficient mice with a C57B6/J background were used. Diet-induced obesity (DIO) C57B6/J mice were fed a 60 kcal% fat diet for 12 weeks prior to performing experiments. After syngeneic hematopoietic stem cell transplantation (HSCT), either TM5614 (a specific inhibitor of PAI-1 molecules) or vehicle (distilled water) were administered daily via oral gavage using a feeding needle for 14 consecutive days. Results: We compared the hematopoietic regeneration after syngeneic HSCT between femurs (microenvironment with a low BMA content) and tail vertebrae (which consist of abundant BMAs). As expected, the microenvironment in tail vertebrae was significantly associated with higher PAI-1 expression in comparison with that in femurs. Hematopoietic regeneration was less efficient in the tail vertebrae, with a reduced number of total donor hematopoietic and CD34-Lin-Sca1+c-kit+ cells observed than in femurs at 1 and 4 weeks following syngeneic HSCT. This impaired regeneration was significantly alleviated in PAI-1-knockout (Figure a) and PAI-1 inhibitor-treated mice (Figure b). Following this, we examined the effects of PAI-1 inhibitor on hematopoietic regeneration after syngeneic HSCT in DIO mice, which had BMA-rich bone marrow microenvironment associated with higher PAI-1 expression compared with that in normal-weight mice. The comparable homing activity of donor cell was observed in normal-weight and DIO mice by using PKH26 staining. However, DIO mice, in comparison to normal-weight mice, exhibited impaired hematopoietic regeneration, with a reduced number of total donor hematopoietic, CD34-Lin-Sca1+c-kit+, and CD48-CD150+Lin-Sca1+c-kit+ SLAM cells at 1 week following HSCT. The impaired hematopoietic regeneration was completely restored by PAI-1 inhibitor administration in these mice (Figure c and d). Furthermore, the number of total donor hematopoietic and CD34-Lin-Sca1+c-kit+ cells at 3 weeks following HSCT was significantly higher in PAI-1 inhibitor treated DIO mice than in normal-weight mice. Conclusion: Our results indicated that PAI-1 hampers hematopoietic regeneration in the BMA-rich microenvironment and the blockade of PAI-1 activity could be a novel therapeutic approach to facilitate hematopoietic regeneration after HSCT in BMA-rich patients. Figure Disclosures Onizuka: Sumitomo Dainippon Pharma: Research Funding; Astellas: Research Funding; Novartis: Research Funding; pfizer: Research Funding; Chugai Pharma: Research Funding; Bristol-Myers Squibb: Research Funding. Ando:Eisai: Research Funding.

Arctigenin alleviates TGF-β1-induced epithelial-mesenchymal transition and PAI-1 expression via AMPK/NF-κB pathway in peritoneal mesothelial cells

Peritoneal fibrosis (PF) caused by long-term peritoneal dialysis is closely associated with the epithelial-mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMCs). Moreover, the anti-fibrotic role of Arctigenin (Arc) has been reported in several fibrosis disorders. Therefore, the preventive effect of Arc on transforming growth factor-β1 (TGF-β1)-induced EMT and the underlying mechanisms in HPMCs was investigated in this study. Firstly, the PD model was established by TGF-β1 stimulation in cultured HPMCs in vitro, we found that TGF-β1 significantly increased the EMT markers (α-SMA, vimentin, and fibronectin) and plasminogen activator inhibitor type 1 (PAI-1) expressions, but decreased epithelial marker (E-cadherin). Co-treatment with Arc (10, 20, 40 μM) ameliorated TGF-β1-induced EMT in a dose-dependent manner, and the expression of PAI-1 was also inhibited by Arc, which was abrogated by restoration of PAI-1. Moreover, Arc enhanced the phosphorylated AMP-activated protein kinase (AMPK), but inhibited the phosphorylated IκBα level and nuclear translocation of nuclear factor κB (NF-κB) p65 in TGF-β1-induced HPMCs. ChIP and Luciferase reporter assays verified that the increased binding capacity of NF-κB to the promoter of PAI-1 induced by TGF-β1 was reversely attenuated by Arc in HPMCs. However, the effect of Arc on TGF-β1-induced NF-κB activation, PAI-1 expression and EMT in HPMCs was attenuated by AMPK agonist Compound C. In conclusion, these data demonstrated that Arc suppressed TGF-β1-induced EMT by activating the AMPK/NF-κB pathway to inhibit PAI-1 expression in HPMCs. Therefore, Arc might act as a potential therapeutic agent for PD treatment.

Effect of Periodontal Surgery on Tissue Plasminogen Activator and Plasminogen Activator Inhibitor Type-1 Gene Expression in Gingival Tissues of Periodontitis Patients: A Controlled Before-And-After Study

Background: Regulation of the plasminogen activation system (PAS) is a vital component in governing proteolytic events within the extracellular matrix (ECM). PAS is believed to play a substantial role in the destruction and healing of periodontal tissues. Thus, the current work aimed to study the histopathological effect of open flap debridement (OFD) on periodontitis, as well as its effect on tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1) gene levels in gingival tissues. Methods: A total of 30 subjects were enrolled in the present study. They were divided into two groups: Group I (control group) included 10 periodontally healthy volunteers and group II (periodontitis group) comprised 20 patients suffering from stage III grade B periodontitis. Gingival tissue samples were collected from all periodontitis patients, before and after OFD, and from healthy controls. Hematoxylin and eosin (H&amp;E) stained slides were subsequently examined and gene expression levels of t-PA and PAI-1 were assessed in the gingiva through quantitative reverse transcription polymerase chain reaction (RT-PCR). Results: Gingival tissue samples from periodontitis patients showed widely dilated blood vessels, diffuse hemorrhage, areas of edema, and disorganized collagen fibers together with large amounts of inflammatory cells in between. Following OFD, smaller sized blood vessels, a restored collagen fiber distribution, and an obvious decrease in the inflammatory infiltrate were noted. Gene expression levels of t-PA and PAI-1 were significantly higher in the periodontitis patients compared to the healthy controls. Although their levels showed a significant decrease following OFD in the periodontitis group, they were still significantly higher than the control group. Conclusion: OFD procedures resulted in down regulation of t-PA and PAI-1 expression levels in the gingiva of periodontitis patients, which could signify an important role of these proteins on periodontal disease progression.

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.

Role of Plasminogen Activator Inhibitor Type 1 in Pathologies of Female Reproductive Diseases.

Normal pregnancy is a state of hypercoagulability with diminishing fibrinolytic activity, which is mainly caused by an increase of plasminogen activator inhibitor type 1 (PAI-1). PAI-1 is the main inhibitor of plasminogen activators, including tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). In human placentas, PAI-1 is expressed in extravillous interstitial trophoblasts and vascular trophoblasts. During implantation and placentation, PAI-1 is responsible for inhibiting extra cellular matrix (ECM) degradation, thereby causing an inhibition of trophoblasts invasion. In the present study, we have reviewed the literature of various reproductive diseases where PAI-1 plays a role. PAI-1 levels are increased in patients with recurrent pregnancy losses (RPL), preeclampsia, intrauterine growth restriction (IUGR), gestational diabetes mellitus (GDM) in the previous pregnancy, endometriosis and polycystic ovary syndrome (PCOS). In general, an increased expression of PAI-1 in the blood is associated with an increased risk for infertility and a worse pregnancy outcome. GDM and PCOS are related to the genetic role of the 4G/5G polymorphism of PAI-1. This review provides an overview of the current knowledge of the role of PAI-1 in reproductive diseases. PAI-1 represents a promising monitoring biomarker for reproductive diseases and may be a treatment target in the near future.

The potential of PAI-1 expression in needle biopsies as a predictive marker for prostate cancer

AbstractThe relative abundance of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) in transurethral resections of the prostate (TURP) has been shown to correlate with disease state. The objective of this study was to assay for uPA and PAI-1 in prostate needle biopsies, and to test their potential as predictive markers for prostate cancer (PCa). uPA and PAI-1 levels were determined for 111 patients (55 PCa; 56 benign prostatic hyperplasia (BPH)), using the FEMTELLE enzyme-linked immunosorbent (ELISA) assay. The PAI-1 concentrations for PCa and BPH patients differed significantly (p = 0.0403) and a level of ≥ 4.5 ng/mg protein in men 60 years and older appears to be predictive of PCa, with a sensitivity of 63%. uPA plays a minor role as a potential marker in biopsy tissue, a feature noted in our recent TURP tissue studies, and elsewhere. The potential utility of the uPA/PAI-1 ratio as a predictor of prostate disease, as previously suggested for TURP tissue, is not app...

Decreased expression of the plasminogen activator inhibitor type 1 is involved in degradation of extracellular matrix surrounding cervical cancer stem cells.

The plasminogen activator (PA) system consists of plasminogen activator inhibitor type 1 (PAI-1), urokinase-type plasminogen activator and its receptor (uPA and uPAR). PAI-1 inhibits the activation of uPA (which converts plasminogen to plasmin), and is involved in cancer invasion and metastasis, by remodeling the extracellular matrix (ECM) through regulating plasmin. Cancer stem cells (CSCs) are a small subset of cells within tumors, and are thought to be involved in tumor recurrence and metastasis. Considering these facts, we investigated the relationship between PAI-1 and cervical CSCs. We used ALDH1 as a marker of cervical CSCs. First, we demonstrated that culturing ALDH1-high cells and ALDH-low cells on collagen IV-coted plates increased their expression of active PAI-1 (ELISA), and these increases were suggested to be at mRNA expression levels (RT-qPCR). Secondly, we demonstrated PAI-1 was indeed involved in the ECM maintenance. With gelatin zymography assays, we found that ALDH1-high cells and ALDH-low cells expressed pro-matrix metalloproteinase-2 (pro-MMP-2) irrespective of their coatings. With gelatinase/collagenase assay kit, we confirmed that collagenase activity was increased when ALDH1-low cells were exposed to TM5275, a small molecule inhibitor of PAI-1. Putting the data together, we hypothesized that cancer cells adhered to basal membrane secrete abundant PAI-1, on the other hand, cancer cells (especially CSCs rather than non-CSCs) distant from basal membrane secrete less PAI-1, which makes the ECM surrounding CSCs more susceptible to degradation. Our study could be an explanation of conflicting reports, where some researchers found negative impacts of PAI-1 expression on clinical outcomes and others not, by considering the concept of CSCs.

Urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) in breast cancer - correlation with traditional prognostic factors.

BackgroundUrokinase plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) play a key role in tumour invasion and metastasis. High levels of both proteolytic enzymes are associated with poor prognosis in breast cancer patients. The purpose of this study was to evaluate the correlation between traditional prognostic factors and uPA and PAI-1 expression in primary tumour of breast cancer patients.Patients and methods.606 primary breast cancer patients were enrolled in the prospective study in the Department of gynaecological oncology and breast oncology at the University Medical Centre Maribor between the years 2004 and 2010. We evaluated the traditional prognostic factors (age, menopausal status, tumour size, pathohistological type, histologic grade, lymph node status, lymphovascular invasion and hormone receptor status), together with uPA and PAI-1. We used Spearman’s rank correlation, Mann Whitney U test and χ2 test for statistical analysis.ResultsOur findings indicate a positive correlation between uPA and tumour size (p < 0.001), grade (p < 0.001), histological type (p < 0.001), lymphovascular invasion (p = 0.01) and a negative correlation between uPA and hormone receptor status (p < 0.001). They also indicate a positive correlation between PAI-1 and tumour size (p = 0.004), grade (p < 0.001), pathohistological type (p < 0.001) and negative correlation between PAI-1 and hormone receptor status (p = 0.002).ConclusionsOur study showed a relationship between uPA and PAI-1 and traditional prognostic factors. Their role as prognostic and predictive factors remains to be further evaluated.

TGF-β1 prevents blood–brain barrier damage and hemorrhagic transformation after thrombolysis in rats

Transforming growth factor-beta1 (TGF-β1) is well known to promote extracellular matrix accumulation. Recent studies demonstrated that TGF-β1 protects against blood–brain barrier (BBB) disruption in the condition of inflammatory pain and stroke. In the present study, we investigated whether TGF-β1 can maintain BBB integrity and prevent hemorrhagic transformation (HT) after recombinant tissue plasminogen activator (rt-PA) treatment in a rat model of thromboembolic middle cerebral artery occlusion (MCAO). Three hours after MCAO, rats were given saline, rt-PA alone or rt-PA combined with TGF-β1 intravenously. Animals were sacrificed 24h after surgery. HT was calculated as hemorrhagic score. Evans blue dye extravasation was measured for BBB disruption. Basement membrane damage was observed by electron microscopy and quantified by collagen IV and laminin immunostaining. Gelatin zymography was used to measure the activities of matrix metalloproteinase (MMP)-2 and MMP-9. Western blot was performed for the expressions of MMP-2, MMP-9 and plasminogen activator inhibitor type-1 (PAI-1). Rats treated with rt-PA showed elevations in basement membrane damage, BBB disruption and HT. These phenomena were reduced in rats treated by TGF-β1. We also showed that TGF-β1 inhibited rt-PA mediated induction of MMP-2 and MMP-9. Meanwhile, TGF-β1 upregulated PAI-1 expression which was reduced by rt-PA. Taken together, these results suggest that TGF-β1 can reduce rt-PA induced basement membrane degradation, BBB disruption and HT. One possible mechanism is associated with the elevation of PAI-1. Suppression of MMP-2 and MMP-9 elevated by rt-PA may be another mechanism contributing to the protective effects of TGF-β1.

Determining the association between adipokine expression in multiple tissues and phenotypic features of non-alcoholic fatty liver disease in obesity.

Objectives:Non-alcoholic fatty liver disease (NAFLD) is an obesity-associated disease, and in obesity adipokines are believed to be involved in the development of NAFLD. However, it is still not clear whether adipokines in the liver and/or adipose tissues can be related to the development of specific characteristics of NAFLD, such as steatosis and inflammation. We aimed to address this question by simultaneously examining the adipokine expression in three tissue types in obese individuals.Methods:We enrolled 93 severely obese individuals with NAFLD, varying from simple steatosis to severe non-alcoholic steatohepatitis. Their expression of 48 adipokines in the liver, visceral and subcutaneous adipose tissue (SAT) was correlated to their phenotypic features of NAFLD. We further determined whether the correlations were tissue specific and/or independent of covariates, including age, sex, obesity, insulin resistance and type 2 diabetes (T2D).Results:The expression of adipokines showed a liver- and adipose tissue-specific pattern. We identified that the expression of leptin, angiopoietin 2 (ANGPT2) and chemerin in visceral adipose tissue (VAT) was associated with different NAFLD features, including steatosis, ballooning, portal and lobular inflammation. In addition, the expression of tumor necrosis factor (TNF), plasminogen activator inhibitor type 1 (PAI-1), insulin-like growth factor 1 (somatomedin C) (IGF1) and chemokine (C-X-C motif) ligand 10 (CXCL10) in the liver tissue and the expression of interleukin 1 receptor antagonist (IL1RN) in both the liver and SAT were associated with NAFLD features. The correlations between ANGPT2 and CXCL10, and NAFLD features were dependent on insulin resistance and T2D, but for the other genes the correlation with at least one NAFLD feature remained significant after correcting for the covariates.Conclusions:Our results suggest that in obese individuals, VAT-derived leptin and chemerin, and hepatic expression of TNF, IGF1, IL1RN and PAI-1 are involved in the development of NAFLD features. Further, functional studies are warranted to establish a causal relationship.

IRAP deficiency attenuates diet-induced obesity in mice through increased energy expenditure

Activation of the adipose renin–angiotensin system contributes to the development of obesity and metabolic syndrome. Insulin-regulated aminopeptidase (IRAP) has been identified a key regulator of GLUT4 transporter as well as angiotensin IV (AngIV) receptor (AT4R). Although AngII-AT1R axis appears as anorexigenic and as an effector of energy expenditure, the impact of AngIV-IRAP/AT4R axis on energy metabolism remains unknown. The aim was to determine the role of IRAP in energy metabolism in mice. Methods and resultsIn adipocyte culture, plasminogen activator inhibitor type 1 (PAI-1) expression levels were diminished in IRAP knockout (IRAP−/−) if compared with those of wild-type (C57Bl/6J, WT) mice. Mice were fed high-fat diet (32% fat) at age of 8weeks. At the entry, body weight, body fat content, and parameters of saccharometabolism were similar between groups. However, IRAP−/− mice exhibited blunted body weight gain compared to that of WT mice, despite comparable food intake and physical activity. At 20weeks of age, IRAP−/− mice had 25% lower body weight than WT mice. Glucose and insulin tolerance tests revealed that the glucose disposal and the hypoglycemic effect of insulin were pronounced in IRAP−/− mice after a high fat diet. Indirect calorimetry demonstrated that whole-body oxygen consumption rates were significantly higher in IRAP−/− mice by 18% with mild hyperthermia. Analysis of brown adipose tissue (BAT) in IRAP−/− showed increased levels of uncoupling protein-1 (UCP-1) at basal level and adaptive thermogenesis was not impaired. ConclusionsIRAP deficiency may lead to suppression of PAI-1 expression in adipocytes and upregulation of UCP-1-mediated thermogenesis in BAT and increased energy expenditure to prevent the development of obesity, and these facts suggest a therapeutic potential of IRAP/AT4R blockade in diet-induced obesity.

Systemic inhibition and liver‐specific over‐expression of PAI‐1 failed to improve survival in all‐inclusive populations or homogenous cohorts of CLP mice

The role of plasminogen activator inhibitor type-1 (PAI-1) in abdominal sepsis remains elusive. To study the influence of inhibition and over-expression of PAI-1 upon survival in cecal ligation and puncture (CLP) sepsis. (i) Mice underwent moderate CLP and received 10 mg kg(-1) of either monoclonal anti-PAI-1 (MA-MP6H6) or control (MA-Control) antibody intravenously at 0, 18 or 30 h post-CLP. The 30-h treatment group was additionally stratified into mice predicted to survive (P-SUR) or die (P-DIE) based on IL 6 measured at 24 h post-CLP. (ii) PAI-1 expression was induced with pLIVE.PAI-1 plasmid administered 72 h pre-CLP. Blood was sampled for 5 days and survival was monitored for 28 days. MA-MP6H6 effectively neutralized active PAI-1 and fully restored fibrinolysis while PAI-1 over-expression was liver-specific and correlated with PAI-1 increase in the blood. Without stratification, MA-MP6H6 co-/post-treatment conferred no survival benefit. Prospective stratification (IL-6 cut-off: 14 ng mL(-1) ) suggested increased mortality by MA-MP6H6 treatment in P-SUR that reached 30% difference (vs. MA-Control; P < 0.05) after a retrospective cut-off readjustment to 3.3 ng mL(-1) for better P-SUR homogeneity. Subsequent prospective anti-PAI-1 treatment in P-SUR mice with 3.3 ng mL(-1) cut-off demonstrated a negative but statistically insignificant effect: mortality was higher by 17% after MA-MP6H6 vs. MA-Control. Over-expression of PAI 1 did not alter post-CLP survival. Neither PAI-1 inhibition nor over-expression meaningfully modified inflammatory response and/or organ function. Restoration of fibrinolysis in early abdominal sepsis was not beneficial and it may prove detrimental in subjects with the lowest risk of death, while preemptive PAI-1 up-regulation at the current magnitude was not protective.

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.

Species-Specific Regulation of t-PA and PAI-1 Gene Expression in Human and Rat Astrocytes.

In recent years, the role and physiological regulation of the serine protease tissue-type plasminogen activator (t-PA) and its inhibitors, including plasminogen activator inhibitor type-1 (PAI-1), in the brain have received much attention. However, as studies focusing these issues are difficult to perform in humans, a great majority of the studies conducted to date have utilized rodent in vivo and/or in vitro models. In view of the species-specific structural differences present in both the t-PA and the PAI-1 promoters, we have compared the response of these genes in astrocytes of rat and human origin. We reveal marked quantitative and qualitative species-specific differences in gene induction following treatment with various physiological and pathological stimuli. Thus, our findings are of importance for the interpretation of previous and future results related to t-PA and PAI-1 expression.

High-density lipoprotein from patients with coronary heart disease loses anti-thrombotic effects on endothelial cells: impact on arterial thrombus formation.

Thrombus formation is determined by the balance between pro- thrombotic mediators and anti-thrombotic factors.High-density lipoprotein (HDL) from healthy subjects exerts anti-thrombotic properties. Whether this is also the case for HDL from patients with stable coronary heart disease (CHD) or acute coronary syndrome (ACS) is unknown.In human aortic endothelial cells in culture,HDL (50 µg/ml) from healthy subjects (HS) inhibited thrombin-induced tissue factor (TF) expression and activity, while HDL (50 µg/ml) from CHD and ACS patients did not. Similarly, only healthy HDL increased endothelial tissue factor pathway inhibitor (TFPI) expression and tissue plasminogen activator (tPA) release, while HDL from CHD and ACS patients had no effect. Healthy HDL inhibited thrombin-induced plasminogen activator inhibitor type 1 (PAI-1) expression, while HDL from ACS patients enhanced endothelial PAI-1 expression. Inhibition of nitric oxide (NO) formation with L-NAME (100 µmol/l) abolished the anti-thrombotic effects of healthy HDL on TF, TFPI, and tPA expression. The exogenous nitric oxide donor, DETANO, mimicked the effects of healthy HDL and counterbalanced the loss of anti-thrombotic effects of HDL from CHD and ACS patients in endothelial cells. In line with this observation, healthy HDL, in contrast to HDL from CHD and ACS patients, increased endothelial NO production. In the laser-injured carotid artery of the mouse, thrombus formation was delayed in animals treated with healthy HDL compared with mice treated with vehicle or HDL from patients with CHD or ACS. In conclusion, HDL from CHD and ACS patients loses the ability of healthy HDL to suppress TF and to increase TFPI and t-PA and instead enhances PAI-1 and arterial thrombus formation.