PAI-1 Activity Research Articles

MiR-421 and miR-30c Inhibit SERPINE 1 Gene Expression in Human Endothelial Cells

In this work, we assessed whether SERPINE1 expression could be under the influence of microRNAs (miRNAs) predicted to bind the SERPINE1 3′UTR region. We specifically focused on the 3′UTR region harboring a common polymorphism, rs1050955, that have been found associated to SERPINE1 monocyte expression, and investigated whether the presence of different alleles at rs1050955 could modify the miRNAs binding efficiency and affect PAI-1 protein levels. We demonstrated that, in human umbilical vein endothelial cells, both miR-421 and miR-30c directly interacted with PAI-1 mRNA to inhibit the expression of the associated protein. However, these inhibitory mechanisms were independent on the allele present at the rs1050955 locus. We further showed that miR-421 levels correlated with PAI-1 activity in the plasma sample of 40 patients with venous thrombosis. Our results strongly suggest that the regulation of PAI-1 molecule could be under the influence of several miRNAs whose measurement in the plasma of patients could be envisaged as a biomarker for inflammatory and thrombotic disorders.

Metformin, but not glimepiride, improves carotid artery diameter and blood flow in patients with type 2 diabetes mellitus

OBJECTIVE:To compare the effects of glimepiride and metformin on vascular reactivity, hemostatic factors and glucose and lipid profiles in patients with type 2 diabetes.METHODS:A prospective study was performed in 16 uncontrolled patients with diabetes previously treated with dietary intervention. The participants were randomized into metformin or glimepiride therapy groups. After four months, the patients were crossed over with no washout period to the alternative treatment for an additional four-month period on similar dosage schedules. The following variables were assessed before and after four months of each treatment: 1) fasting glycemia, insulin, catecholamines, lipid profiles and HbA1 levels; 2) t-PA and PAI-1 (antigen and activity), platelet aggregation and fibrinogen and plasminogen levels; and 3) the flow indices of the carotid and brachial arteries. In addition, at the end of each period, a 12-hour metabolic profile was obtained after fasting and every 2 hours thereafter.RESULTS:Both therapies resulted in similar decreases in fasting glucose, triglyceride and norepinephrine levels, and they increased the fibrinolytic factor plasminogen but decreased t-PA activity. Metformin caused lower insulin and pro-insulin levels and higher glucagon levels and increased systolic carotid diameter and blood flow. Neither metformin nor glimepiride affected endothelial-dependent or endothelial-independent vasodilation of the brachial artery.CONCLUSIONS:Glimepiride and metformin were effective in improving glucose and lipid profiles and norepinephrine levels. Metformin afforded more protection against macrovascular diabetes complications, increased systolic carotid artery diameter and total and systolic blood flow, and decreased insulin levels. As both therapies increased plasminogen levels but reduced t-PA activity, a coagulation process was likely still ongoing.

Blood biomarkers and measures of pulmonary function–A study from the Swedish twin registry

There is great need of biomarkers for research and clinical purposes in COPD. This study explored the relationships between ten putative plasma biomarkers of COPD and physiological measures of reduced lung function. FEV(1), FVC, residual volume/total lung capacity (RV/TLC) and CO diffusion capacity (D(L)CO) were assessed in 357 subjects from the Swedish Twin Registry. The lung function measures were studied in relation to plasma levels of desmosines, C-reactive protein (CRP), plasminogen inhibitor activator (PAI-1) concentration and activity, tissue inhibitor of metalloproteinase (TIMP-1), clara cell protein 16 (CC16), surfactant protein D (SPD), matrix metalloproteinase 9 (MMP-9), hepatocyte growth factor (HGF) and interleukin (IL)-8. After adjustments for age, sex, height, BMI and smoking, FEV(1) was significantly associated with PAI-1 activity and desmosines. RV/TLC was significantly associated with CC16, PAI-1 concentration and PAI-1 activity, and D(L)CO was significantly associated with desmosines, TIMP-1 and CRP. When the multivariate analysis was restricted to subjects with COPD (i.e., FEV(1)/FVC < 0.70), CRP and desmosines were inversely associated with lung function. Several biomarkers were associated with lung function in this cross-sectional study. Especially CRP and desmosines could be useful markers to assess disease severity in subjects with COPD.

Serum 25-hydroxyvitamin D concentration, established and emerging cardiovascular risk factors and risk of myocardial infarction before the age of 60 years

ObjectiveWe examined the relationships of serum 25-hydroxyvitamin D (25(OH)D) concentration to established and emerging cardiovascular risk factors and risk of myocardial infarction (MI) in a population-based case–control study of MI before the age of 60 years. MethodsA total of 387 survivors of a first MI and 387 sex- and age-matched controls were included. Fasting blood samples drawn three months after the MI in cases and at the same time in the matched controls were used for biochemical analyses. ResultsSerum concentrations of 25(OH)D, adjusted for seasonal variation, were lower in cases than controls (55.0 (40.0–71.0) nmol/L vs 60.5 (47.0–75.0) nmol/L; median (interquartile range); standardized odds ratio (OR) for MI with 95% confidence interval in univariable analysis: 0.80 (0.69–0.93); p = 0.003). The 25(OH)D association with MI disappeared after adjustment for established and emerging risk factors (OR: 1.01 (0.82–1.25)). Current smoking and plasma levels of proinsulin and PAI-1 activity were independently associated with 25(OH)D in controls, whereas waist circumference, plasma triglycerides, proinsulin, PAI-1 activity and cystatin C, and non-Nordic ethnicity were independently associated with 25(OH)D in patients. Serial measurements of 25(OH)D (samples drawn <4 h and 3 months after the onset of MI) in 57 patients showed no systematic differences between sampling times. ConclusionVitamin D insufficiency, which is associated with a multitude of metabolic, procoagulant and inflammatory perturbations, is not independently related to premature MI. This suggests that vitamin D insufficiency either constitutes an epiphenomenon or increases the risk of MI by promoting established risk factor mechanisms that predispose to atherothrombosis.

Abstract 5187: Urokinase plasminogen activator receptor (uPAR) and integrin α5β1 regulate plasminogen activator inhibitor-1 (PAI-1) activity in medulloblastoma cells

Abstract The urokinase plasminogen activator (uPA) system is a dynamic extracellular protease system that regulates both proteolytic and non-proteolytic events associated with cancer progression. In earlier studies, we have demonstrated that radiation-induced cell adhesion was associated with uPAR expression and downregulation of uPAR effectively inhibited radiation-induced cell adhesion of medulloblastoma cells. Herein, we further investigated the impact of uPAR downregulation on plasminogen activator inhibitor (PAI-1), a key regulator component of the uPA system, and its role in cell adhesion. With radiation treatment, the levels of uPAR and PAI-1 increased in D283 and UW228 cells. Surprisingly, knockdown of uPAR elevated the levels of PAI-1. The increase in PAI-1 levels in uPAR-knocked down cells was regulated by activation of cJUN/CREB signaling molecules. Moreover, studies show that the reduced interaction between uPAR and integrin α5β1 plays a critical role in PAI-1 release into the extracellular matrix, thereby enhancing the cell detachment process. Incubation of medulloblastoma cells with Tiplaxtinin-PAI-039, a potent PAI-1 activity inhibitor, increased the association of uPAR with integrin α5β1 and cell adhesion. Interestingly, prolonged inhibition of PAI-1 activity adversely affected cell viability; we were able to revert this effect by exogenously supplementing cells with recombinant PAI-1. Moreover r-PAI-1 showed no significant effect on cell adhesion whereas it increased the association of uPAR with integrin α5β1. Expression of full-length (FL) uPAR elevated uPA and PAI-1 levels both at the transcriptional and the translational levels. These increased PAI-1 levels play a pivotal role in regulating uPA activity by binding and translocating integrin α5β1 towards the uPA/uPAR complex. Taken together, the results of the present study lead us to conclude that levels of uPAR and integrin α5β1, and their association on the cell surface dictate the function of PAI-1 to maintain extracellular homeostasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5187. doi:1538-7445.AM2012-5187

BMP-9 induces both smad1/5/8 and Smad2/3 phosphorylation, and specific response genes, in chondrocytes

Background: Osteoarthritis is characterized by loss of articular cartilage. The main cartilage degrading enzyme is Matrix metalloproteinase 13 (MMP13). Expression of MMP13 is positively correlated with Activin receptor-like kinase 1 (ALK1) expression. Moreover, terminal differentiation of chondrocytes is induced by ALK1 via phosphorylation of Smad1/5/8 (Smad1/5/8p). BMP-9, also known as GDF-2, has recently been characterized as a potent, high-affinity ALK1 ligand, that is present in serum in high amounts. We wanted to further investigate intracellular BMP-9 signaling in chondrocytes to clarify its role in cartilage maintenance and degradation. Purpose: To study Smad1/5/8 and Smad2/3 phosphorylation kinetics in response to addition of exogenous BMP-9, to investigate BMP-9’s potency on chondrocytes, and to investigate downstream signaling responses on mRNA level. Methods: Both primary bovine chondrocytes, isolated from the metacarpophalangeal joint of 2 year old animals, as well as the murine H4 chondrocyte cell line were used in this study. Chondrocytes were cultured to near confluency, and subsequently incubated with BMP-9. After short term stimulation (1 h), Smad phosphorylation was analyzed by specific Smad2/3p and Smad1/5/8p staining of Western blots. After long term stimulation (24-48 h), expression of PAI-1, a downstream marker of Smad2/3 signaling, and ID-1, a downstream marker of Smad1/5/8 signaling, were analyzed by quantitative real time PCR (qPCR). Results: In both the murine cell line, as well as the primary bovine chondrocytes, addition of BMP-9 resulted in phosphorylation of Smad 1/5/8, that was maximal at very low concentrations (100 pg / ml). Unexpectedly, Smad2/3p was also observed at this concentration. Further investigations showed that BMP-9-induced Smad2/3p was maximal after 1 h, at the physiological relevant concentration of 5 ng/ml. Exposure of chondrocytes to BMP9 not only resulted in both Smad1/5/8p and Smad2/3p but also in transcription of pathway specific response genes, as observed by a significant increase in PAI-1 and ID-1 mRNA levels after both 24 and 48h. In both cell types, collagen type 2A1 and aggrecan mRNA levels were also up regulated by BMP9. In the described experiments, no significant changes in MMP13 expression were observed after 48 h of BMP-9 stimulation. Conclusion: In chondrocytes, BMP9 is capable of inducing both Smad1/5/8p and Smad2/3p. By doing so it can up regulate both PAI-1 and ID-1 simultaneously. BMP-9 is very potent in inducing Smad phosphorylation, as very low concentrations (pg/ml) are sufficient for signal induction. The induction of Smad2/3p and subsequent PAI-1 activity sets BMP-9 apart from other BMPs, like BMP-2 and BMP-6, that only induce Smad1/5/8 phosphorylation. The lack of MMP13 expression after BMP-9 stimulation can be explained by the induced Smad2/3p expression, as this potently blocks MMP13 transcription. Based on these results we propose that exposure of young cartilage to BMP-9 does not result in MMP13 expression due to its ability to induce Smad2/3p. However, we previously reported that in ageing cartilage the ability to phosphorylate Smad2/3 is reduced, and therefore BMP-9 exposure will most likely result in MMP13 production in old cartilage.

Intracoronary delivery of DNAzymes targeting human EGR‐1 reduces infarct size following myocardial ischaemia reperfusion

Despite improvements in treatment, myocardial infarction (MI) remains an important cause of morbidity and mortality. Inflammation arising from ischaemic and reperfusion injury is a key mechanism which underpins myocardial damage and impairment of cardiac function. Early growth response-1 (Egr-1) is an early immediate gene and a master regulator that has been implicated in the pathogenesis of ischaemia-reperfusion (IR) injury. This study sought to examine the effect of selective inhibition of Egr-1 using catalytic deoxyribonucleic acid molecules (DNAzymes, DZs) delivered via the clinically relevant coronary route in a large animal model of myocardial IR. It was hypothesized that Egr-1 inhibition with intracoronary DZ would reduce infarction size by modulating its downstream effector molecules. Egr-1 DZs inhibited the adherence of THP-1 monocytes to IL-1β-activated endothelial cells in vitro and retained its catalytic activity up to 225 min after in vivo administration. In a porcine model of myocardial IR (45 min ischaemia/3 h reperfusion), DZ was taken up in the cytoplasm and nuclei of cardiomyocytes and endothelial cells in the myocardium after intracoronary delivery. Egr-1 DZs reduced infarct size and improved cardiac functional recovery following intracoronary delivery at the initiation of IR in this large animal model of MI. This was associated with inhibition of pro-inflammatory Egr-1 and ICAM-1 expression, and the reduced expression of TNF-α, PAI-1, TF, and myocardial MPO activity in tissue derived from the border zone of the infarct. Taken together, these data suggest that strategies targeting Egr-1 via the intracoronary route after IR injury in pigs have potential therapeutic implications in human MI.

A Plasminogen Activator Inhibitor-1 Inhibitor Reduces Airway Remodeling in a Murine Model of Chronic Asthma

We previously reported that plasminogen activator inhibitor (PAI)-1 deficiency prevents collagen deposition in the airways of ovalbumin (OVA)-challenged mice. In this study, we explored the therapeutic utility of blocking PAI-1 in preventing airway remodeling, using a specific PAI-1 inhibitor, tiplaxtinin. C57BL/6J mice were immunized with intraperitoneal injections of OVA on Days 0, 3, and 6. Starting on Day 11, mice were challenged with phosphate-buffered saline or OVA by nebulization three times per week for 4 weeks. Tiplaxtinin was mixed with chow and administered orally from 1 day before the phosphate-buffered saline or OVA challenge. Lung tissues were harvested after challenge and characterized histologically for infiltrating inflammatory cells, mucus-secreting goblet cells, and collagen deposition. Airway hyperresponsiveness was measured using whole-body plethysmography. Tiplaxtinin treatment significantly decreased levels of PAI-1 activity in bronchoalveolar lavage fluids, which indicates successful blockage of PAI-1 activity in the airways. The number of infiltrated inflammatory cells was reduced by tiplaxtinin treatment in the lungs of the OVA-challenged mice. Furthermore, oral administration of tiplaxtinin significantly attenuated the degree of goblet cell hyperplasia and collagen deposition in the airways of the OVA-challenged mice, and methacholine-induced airway hyperresponsiveness was effectively reduced by tiplaxtinin in these animals. This study supports our previous findings that PAI-1 promotes airway remodeling in a murine model of chronic asthma, and suggests that PAI-1 may be a novel target of treatment of airway remodeling in asthma.

Fibrinolytic status in acute coronary syndromes: Evidence of differences in relation to clinical features and pathophysiological pathways

Limited data are available on the role of innate fibrinolysis in acute coronary syndromes (ACS). In the present study we evaluated the dynamic alterations of fibrinolytic markers in patients presenting with ACS. Tissue-type-(tPA) and urokinase type-(uPA) plasminogen activators, plasminogen activator inhibitor (PAI-1) antigen and activity and thrombin activatable fibrinolysis inhibitor (TAFI) were analysed in 50 patients with ST elevation myocardial infarction (STEMI), 47 non-STEMI patients (NSTEMI), 40 patients with stable coronary artery disease (CAD) and 39 controls. The parameters were measured on day 1 and days 3, 7 and 30. Counts of monocyte subsets, monocyte-platelet aggregates and plasma inflammatory cytokines were assessed on admission. On day 1, TAFI was higher in NSTEMI vs. STEMI (p<0.001) while PAI-1 activity was higher in STEMI (p<0.001). In STEMI, uPA activity levels was low on day 1 but significantly increased on day 30 (p<0.001). TAFI levels were increased in NSTEMI on day 1 and gradually reduced by day 30 (p<0.05). In STEMI, TAFI levels peaked at day 7 (p<0.05) and dropped significantly by day 30 (p<0.05). CD14++CD16+ monocytes were independently associated with PAI-1 activity in ACS (p=0.03). Monocyte-platelet aggregates rather than platelet-free monocytes were an independent determinant of tPA, PAI-1 antigen and TAFI on a multivariate analysis (p<0.05). There are significant differences in fibrinolytic activity between patients with STEMI and NSTEMI. These changes could reflect the role of these factors in post-MI myocardial healing. Monocyte-platelet interactions are independently associated with the regulation of the fibrinolytic status in ACS.

Hormonal regulation of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 gene expression in cultured mouse Sertoli cells.

We have demonstrated for the first time that (i) mouse Sertoli cells predominantly secrete tPA under the action of FSH and cAMP-generating agents, whereas Leydig cells mainly produce uPA; (ii) Sertoli cells are also capable of secreting PAI-1, as well as FSH, growth factors and GnRH increase PAI-1 gene expression; (iii) the increases in tPA and PAI-1 activities by different hormones in the conditioned media of Sertoli cells correspond to the increases in the levels of tPA and PAI-1 mRNA in the cultured cells, suggesting that the synthesis of the activator-inhibitor in mouse Sertoli cells is regulated at a transcriptional level and the tPA secreted by Sertoli cells may be involved in the spermatogenesis.

Effect of activated human protein C on disseminated intravascular coagulation induced by lipopolysaccharide in rats.

Protein C is the zymogen of an anticoagulant serine protease and is converted to its active form (activated protein C: APC) by thrombin in the presence of thrombomodulin. APC plays an important role in regulating coagulation and fibrinolysis by inactivating not only blood coagulation factors Va and VIIIa but also type-1 plasminogen activator inhibitor (PAI-1). The aim of the present study was to examine the effect of a human APC product (designated as CTC-111), compared with that of heparin, on the disseminated intravascular coagulation (DIC) induced by lipopolysaccharide (LPS) in rats. LPS (1 mg/kg/h) infusion was performed through a femoral vein for 4 h. One-fifth amount of the total dosage of CTC-111 or heparin was injected into the other femoral vein, followed by a 4-h infusion of the remainder. Both CTC-111 (10,000-100,000 U/kg) and heparin (400-800 IU/kg) inhibited the decrease in platelet count and fibrinogen level equally. The prolonged activated partial thromboplastin time and prothrombin time observed in DIC rats were further elongated in both CTC-111- and heparin-treated rats. But, this prolongation was less in CTC-111-treated rats than in the heparin-treated ones. Heparin inhibited the increase in fibrin and fibrinogen degradation products more prominently than CTC-111. On the other hand, CTC-111 strongly inhibited the increase in PAI-1 activity but heparin did not. These results suggest that CTC-111 may enhance fibrinolysis through its direct inhibitory effect on PAI-1. The parameters for liver or renal damage, i.e., plasma glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), creatinine (Cre) and blood urea nitrogen (BUN), were significantly increased by LPS infusion. Both CTC-111 (100,000 U/kg) and heparin (800 IU/kg) decreased the increase in GOT and GPT levels significantly, whereas neither affected the increase in Cre or BUN. From these results, the activation of the blood coagulation system might partially contribute to the progression of liver damage caused by LPS, and might be less involved in the progression of renal damage in this model. In conclusion, CTC-111 showed both anticoagulant and profibrinolytic activity in the LPS-induced DIC model without excessive prolongation of coagulation time. From these results, CTC-111 is expected to be a useful remedy for DIC without the risk of bleeding.

Relations of growth-differentiation factor-15 to biomarkers reflecting vascular pathologies in a population-based sample of elderly subjects

Background. Growth-differentiation factor-15 (GDF-15) has recently emerged as a risk predictor in patients with cardiac diseases. GDF-15 is commonly related to cardiovascular risk factors, inflammatory activity and cardiac abnormalities. However, it is not clear whether it might be an indicator of vascular pathologies as well. Methods. Circulating levels of GDF-15 were measured in 1004 elderly community dwellers participating in the PIVUS study. The relations of GDF-15 to biomarkers of endothelial activation (E-selectin, P-selectin, ICAM-1, VCAM-1), extracellular matrix degradation (MMP-9, TIMP-1), coagulatory activity (D-dimer, von Willebrand factor, prothrombin fragment 1 + 2, factor VIIa), and fibrinolytic activity (PAI-1 activity, tPA-antigen) were assessed by multiple linear regressions. Results. The median GDF-15 level was 1135 ng/L. By linear correlation analysis, GDF-15 exhibited a moderate relation to von Willebrand factor (r = 0.30), and weak, albeit significant relations (r = 0.13–0.29) to E-selectin, P-selectin, ICAM-1, VCAM-1, MMP-9, TIMP-1, D-dimer, PAI-1 activity and tPA-antigen. The relations to the assessed biomarkers of endothelial activation, TIMP-1, D-dimer and von Willebrand factor remained significant applying multiple linear regression models adjusted for clinical covariates and echocardiographic data. There were no significant relations between GDF-15 and biomarkers solely reflecting coagulatory activity. Conclusions. In the elderly, GDF-15 reflects endothelial activation and vascular inflammation and thus, multiple pathways involved in the development and progression of atherosclerosis.

Role for Gβγ G-proteins in protease regulation during remodeling of the murine femoral artery

BackgroundIntimal hyperplasia remains the principal lesion in the development of restenosis after vessel wall injury. G-protein coupled receptors are involved in smooth muscle cell proliferation but the role of Gβγ in arterial intimal hyperplasia has not been well defined. The aim of this study is to characterize the expression of Gβγ G-proteins in the developing intimal hyperplasia in a murine model and the impact of disruption of Gβγ signaling on intimal hyperplasia development. MethodsThe murine femoral wire injury model was employed. Specimens were perfusion-fixed and sections were stained with H&E and Movat's stains such that morphometry could be performed using an Image-Pro system. Additional specimens of femoral artery were also harvested and snap frozen for Western blotting for the Gβγ expression and for Western blotting and zymography to allow for the study of gelatinase and plasminogen activator expression and activation. Contralateral vessels were used as controls. Additional vessels were immersed in pluronic gel containing an adenovirus with the Gβγ inhibitor βARKCT. ResultsThe injured femoral arteries developed intimal hyperplasia, while sham vessels did not produce such a response. Cell proliferation peaked at 3-5 d and cell migration at 7 d after injury. There was a marked time-dependent increase in Gβγ over the 28 d following injury. Inhibition of Gβγ with βARKCT inhibited cell proliferation, cell migration and the development of intimal hyperplasia. Inhibition of Gβγ decreased peak uPA activity and expression without increasing early PAI-1 activity and expression. Inhibition of Gβγ reduced peak MMP-2 activity at d 1 but not at d 7 and also reduced peak MMP-9 activity at d 3. Protein expression for both MMP-2 and MMP-9 was also transiently decreased. There were no changes in TIMP-1 and TIMP-2 expression and activity. ConclusionsThese data demonstrate a time-dependent increase in Gβγ G-protein expression following wire injury in the mouse. Inhibition of Gβγ alters cell proliferation and migration with associated changes in MMP-2, MMP-9, and uPA expression and activity.

Effect of orange juice and beverage with phytosterols on cytokines and PAI-1 activity

Inflammation is pivotal in all phases of atherosclerosis. Dietary options which lower inflammatory biomarkers would be an attractive strategy to reduce risk from cardiovascular diseases and cancer. Indeed, fruit and vegetable intake or fruit juice consumption is associated with health and wellness. However, there is a paucity of data examining the effect of orange juice on biomarkers of inflammation in healthy volunteers. We have previously conducted the first placebo-controlled randomized studies examining the effect of sterol fortified orange juice or sterol fortified reduced calorie orange juice beverage supplementation (2g sterols/day) compared to Placebo OJ or Placebo OJBev, and showed significant benefits on the lipid profile as well as significant reduction in hsCRP, the prototypic marker of inflammation and a cardiovascular risk marker. The aim of this study was to examine the effect of orange juice (OJ) or OJ beverage (Bev) alone and fortified with plant sterols (1g/240ml juice or beverage twice a day) on pro-inflammatory cytokines and PAI-1, a marker of impaired fibrinolysis in healthy human volunteers. In the first study, 72 healthy human volunteers received Placebo OJ or Sterol OJ and in the second study, 72 volunteers received OJBev or Sterol OJBev for 8 weeks and blood was drawn at baseline and following supplementation for 8 weeks. Biomarkers of Inflammation (IL-1b, IL-6, TNF, IL-8, IL-10) were assessed in serum using the BD Human Inflammatory Cytokine Cytometric Bead Array and PAI-1 activity was assessed in citrated plasma. OJ or OJBev alone failed to result in any significant effects on circulating cytokine levels or PAI-1 activity. There was a significant reduction in IL-1b with sterol fortified OJ (p<0.05) compared to baseline. In addition, both sterol fortified OJ as well as sterol fortified OJBev resulted in significant reductions in serum IL-6 levels (p<0.01). Thus, sterol fortified OJ and OJ Beverage are able to effectively lower biomarkers of inflammation in healthy human volunteers in addition to providing lipid profile benefits and may thus contribute to decreasing cardiovascular risk.

Regulation of tissue plasminogen activator/plasminogen activator inhibitor‐1 by hydrocortisone in rat primary astrocytes

Although originally known as a plasma serine protease involved in clot dissolution, tPA and its primary inhibitor, PAI-1, play crucial roles in synaptic reorganization and plasticity in the central nervous system. In contrast to the wide array of work conducted in neural cells, relatively little is known about the regulatory mechanism governing tPA/PAI-1 expression in astrocytes. Glucocorticoids (GCs) such as hydrocortisone regulate the expression of tPA/PAI-1 in various biological systems in a tissue-specific manner. However, little is known about GC-mediated regulation of tPA/PAI-1 system in CNS. The aims of the present study were to investigate whether tPA/PAI-1 expression is regulated by hydrocortisone in rat primary astrocytes. Enzyme activity of tPA was decreased in a concentration-dependent manner by hydrocortisone treatment, and the activity of PAI-1 was increased by hydrocortisone. Hydrocortisone did not affect the level of tPA mRNA, which suggests that transcriptional down-regulation of tPA mRNA is not involved in the down-regulation of tPA enzyme activity in astrocytes. However, the level of PAI-1 mRNA and protein was increased. Both hydrocortisone and a tPA-Stop treatment prevented glutamate-induced neurotoxicity in rat cortical primary mixed astrocyte-neuron culture, which suggests a neurotoxic role for tPA in our culture system. Interestingly, hydrocortisone further increased LPS-induced up-regulation of PAI-1 while inhibiting the up-regulation of iNOS and COX-2 expression. Our data show that hydrocortisone up-regulated PAI-1 expression along with down-regulation of tPA activity in both normal and inflammatory conditions.

Characterization of a panel of monoclonal antibodies toward mouse PAI-1 that exert a significant profibrinolytic effect in vivo

IntroductionPAI-1 is the main physiological inhibitor of t-PA and u-PA. Elevated PAI-1 levels have been implicated in the pathogenesis of several thrombotic and non-thrombotic diseases. The effect of PAI-1 inhibition can be studied in mouse models, when appropriate immunological tools are available. The majority of the available monoclonal antibodies against PAI-1 have been raised against human PAI-1. Even though some of these antibodies cross-react with non-glycosylated PAI-1 from different species, these antibodies often do not cross-react sufficiently with glycosylated mouse PAI-1. Moreover, the antibodies that cross-react with glycosylated mouse PAI-1 often have decreased inhibitory properties in the presence of vitronectin. Our objective was the generation of a panel of monoclonal antibodies reacting with vitronectin-bound glycosylated mouse PAI-1. ResultsFive monoclonal antibodies revealed binding to glycosylated mouse PAI-1 and exerted a strong (i.e. 58-80% inhibition of PAI-1 activity) inhibitory effect toward mouse PAI-1. Similar inhibitory effects were seen in the presence of a 33-fold molar excess of vitronectin. The PAI-1 inhibitory potential of the antibodies in vivo was demonstrated in a thromboembolism model, in which the evaluated antibodies significantly increased the percentage of mice with normal physical activity in comparison to mice treated with negative control antibody. ConclusionsTo the best of our knowledge this is the first panel of monoclonal antibodies that can inhibit mouse PAI-1 in the presence of vitronectin and that show a profibrinolytic effect in vivo. Therefore these antibodies provide excellent immunological tools to further investigate the role of PAI-1 in mouse models.

Markers of fibrinolysis as predictors for maintenance of sinus rhythm after electrical cardioversion

IntroductionInflammation, endothelial dysfunction and metabolic pathways provide possible links between the inflammatory and hypofibrinolytic states in atrial fibrillation. Our aim was to explore the role of mass concentrations of PAI-1 and tPA, activities of PAI-1 and tPA as predictors of recurrence of atrial fibrillation adjusted for CRP. Materials and methodsThe study included 129 patients with persistent atrial fibrillation. Laboratory analyses were performed including PAI-1 activity, PAI-1 mass, tPA activity, tPA mass and CRP in baseline. Patients were then randomized to atorvastatin (40mg, two tablets once daily) or placebo, initiated at least 14days before the elective cardioversion. Further samples and follow-up were made at day 2 and 30days after cardioversion. ResultsIn univariate logistic regression no fibrinolytic variable was significantly correlated with rhythm in day 30. In multivariate analysis lower PAI-1 mass was significantly associated with sinus rhythm in all models including fibrinolytic variables, CRP, metabolic components, age, hypertension and smoking. After adding treatment allocation to the fully adjusted model, PAI-1 mass remained significantly associated with sinus rhythm both at day 2 and 30 (OR 0.98; 95% CI 0.95–1.00). ConclusionsNo fibrinolytic component alone was found to be a predictor of recurrence of atrial fibrillation. In multivariate models lower PAI-1 mass was associated with sinus rhythm even after adjusting for CRP, markers of the metabolic syndrome and treatment with atorvastatin. Our findings suggest a patophysiological link between AF and PAI-1 mass but the relation to inflammation remains unclear.

Ischemic stroke in patients with type 2 diabetes: Relationship between decreased insulin sensitivity and fibrinolysis impairment

The role of insulin sensitivity (IS), as well as the association of IS with fibrinolysis impairment, in the occurrence of ischemic stroke, has not been clarified. The study was aimed to analyze IS, plasma insulin (PI) and plasminogen activator inhibitor (PAI)-1 levels in 34 type 2 diabetics (T2D) with ischemic stroke (group A), 30 T2D without ischemic stroke (group B), 33 nondiabetics with ischemic stroke (group C) and 33 healthy controls (group D). Ischemic stroke was confirmed by clinical and neuroimaging criteria. IS levels were determined by the minimal model analysis (Si index). Plasma insulin levels were measured by radioimmunoassay and PAI-1 activity was performed by the plasminogen chromogenic plasmin substrate assay. We found that Silevels were significantly lower in group A vs. B (1.17+/-0.66 vs. 2.79+/-0.62 min-1/mU/Lx104; p<0.001) and in C vs. D (3.25+/-0.84 vs. 6.03+/-1.69 min-1/mU/Lx104; p<0.001), while PI levels were higher in group A vs. B (19.46+/-4.11 vs. 14.79+/-1.75 mU/L; p<0.001) and in C vs. D (15.16+/-2.23 vs. 7.54+/-2.03 mU/L; p <0.001). Also, PAI-1 activity was significantly higher in group A vs. B (7.78+/-1.05 i 4.56+/-0.71 mU/L; p<0.001) and in C vs D (4.65+/-0.69 i 3.48+/-1.29 mU/L; p<0.001). Moreover, Silevels correlated with PAI-1, both in T2D and nondiabetics. Our results indicate that appearance of ischemic stroke was associated with decreased insulin sensitivity, together with compensatory hyperinsulinemia, both in T2D and nondiabetics. Our results imply that impaired insulin sensitivity exerts its atherogenic influence, at least in part, by decreased fibrinolysis.

Whole grain wheat sourdough bread does not affect plasminogen activator inhibitor-1 in adults with normal or impaired carbohydrate metabolism

Background and aimsEpidemiological studies suggest whole grain consumption is associated with a reduced risk of cardiovascular disease (CVD), possibly through alterations in glucose metabolism and subsequent effects on plasminogen activator inhibitor (PAI)-1, a novel biomarker for CVD. Our aim was to investigate the effect of 6 wk of whole grain wheat sourdough bread consumption versus refined white bread on PAI-1. Methods and ResultsNormoglycemic/normoinsulinemic (NGI; n = 14; age 53 ± 6 y; BMI 26.5 ± 2.9 kg/m2) and hyperglycemic/hyperinsulinemic (HGI; n = 14; age 57 ± 7 y; BMI 35.7 ± 5.7 kg/m2) adults incorporated whole grain wheat sourdough (162.5 g) or white (168.8 g) bread into their diet, for 6 wk in a randomized crossover study. Pre- and post-intervention, fasting blood samples were analyzed for PAI-1 (primary outcome), as well as glucose, insulin and glucagon (secondary outcomes) at fasting and postprandially after an oral glucose tolerance test (OGTT). Anthropometric measures, fasting glucose, insulin, glucagon and PAI-1 antigen and activity were not different between treatments in either NGI or HGI adults. Glucose incremental area under the curve (iAUC) was lower (19%, P = 0.02) after 6 wk consumption of whole grain wheat sourdough bread compared to white bread in the HGI group, with no differences in insulin or glucagon iAUC in either group. ConclusionOur data showed decreased glucose iAUC after an OGTT following 6 wk whole grain wheat bread consumption in adults with differing glycemic/insulinemic status, but no improvements in PAI-1 or fasting glycemic parameters.

E0147 Mechanism of different activators of PPARαregulating plasminogen activator inhibitor-1 expression

ObjectiveTo observe the effects of fenofibrate and linoleic acid which was the different activators of PPARα on the expression of PAI-1 and PPARα in HepG2 cells. To investigate the relationship...