Plasminogen Activator Inhibitor-1 Protein Levels Research Articles

Plasminogen activator inhibitor-1 serum levels in frontotemporal lobar degeneration.

Plasminogen activator inhibitor-1 (PAI-1) impedes brain plasmin synthesis. Reduced plasmin activity facilitates cumulation of amyloid beta (Aβ) in Alzheimer's disease (AD). Since plasmin also regulates the synaptic activity, it is possible that altered PAI-1 is present in other neurodegenerative disorders. We investigated whether PAI-1 and its counter-regulatory tissue plasminogen activator (tPA) are altered in serum of patients with dementia due to frontotemporal lobar degeneration (FTLD). Thirty five FTLD patients (21 in mild cognitive impairment stage (MCI) and 14 in dementia stage) and 10 cognitively healthy controls were recruited. Serum tPA and PAI-1 protein levels were measured by anova. Correlation between biochemical and demographic data were explored by measuring Pearson correlation coefficient. Serum PAI-1 levels were elevated in the FTLD dementia group as compared to FTLD MCI and controls. tPA serum levels and PAI-1/tPA ratio did not significantly differ among groups. There was a negative correlation between PAI-1 serum levels and disease severity measured by MMSE score. No correlations of tPA serum levels and PAI-1/tPA ratio with MMSE were found. Increased PAI-1 serum levels may serve as a marker of dementia in FTLD, suggesting that, besides Aβ pathway, the plasmin system may affect cognition through synaptic activity.

P184 Identification of the PAI-1, as a potential non-invasive, fecal biomarker in the Inflammatory Bowel Diseases

Abstract Background Inflammatory bowel diseases (IBD) are defined by unregulated immune response leading to intestinal inflammation. Monitoring of the intestinal inflammation 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 and therapeutic response, will have important clinical implications. Our previous cytokine profiling identified that plasminogen activator inhibitor 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 Biopsy, serum and fecal samples were collected from more than 150 active IBD patients and controls without IBD. Inclusion criteria included change of therapy or initiation of new treatment for IBD patients. Immunofluorescence (IF) staining was used to observe the presence and localisation of cytokines. Total protein and mRNA were isolated from biopsies and fecal samples. Protein levels were measured by ELISA and gene expression by qRT-PCR. Results The IF staining of the biopsy samples revealed a significantly higher percentage of PAI-1 positive cells in the inflamed mucosa compared to the control. The relative mRNA expression of PAI-1 was significant higher in non-responders and untreated, inflamed IBD patients compared to controls and responders. On the other hand, we were not able to detect significant changes between responders and non-responders in gene expression of TNF-α, IL-1β and IL-6. Serum, mucosal and fecal PAI-1 protein levels were significantly higher in IBD patients compared to controls. Whereas no significant difference was observed between Crohn’s disease and ulcerative colitis patients. The PAI-1 level was significant increased in endoscopically and clinically active disease compared to inactive patients and controls. In responders, PAI-1 concentration significantly decreased after initiation of therapy compared to non-responders. Significantly upregulated fecal PAI-1 level was found in active IBD compared to patients with inactive IBD, tumor, adenoma, diverticulosis and control. In addition, the stability analysis of fecal PAI-1 showed that the protein was detectable up to one week at room temperature and 4ºC. Conclusion Our findings suggest that serum, mucosal and fecal PAI-1 expression correlates with disease activity in IBD patients, suggesting that it could be utilized to monitor disease activity and possibly therapy response as a non-invasive biomarker in IBD.

Visceral fat-specific regulation of plasminogen activator inhibitor-1 in aged septic mice.

Elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1) are documented in patients with sepsis and levels positively correlate with disease severity and mortality. Our previous work demonstrated that visceral adipose tissues (VAT) are a major source of PAI-1, especially in the aged (murine endotoxemia), that circulating PAI-1 protein levels match the trajectory of PAI-1 transcript levels in VAT (clinical sepsis), and that PAI-1 in both VAT and plasma are positively associated with acute kidney injury (AKI) in septic patients. In the current study utilizing preclinical sepsis models, PAI-1 tissue distribution was examined and cellular sources, as well as mechanisms mediating PAI-1 induction in VAT, were identified. In aged mice with sepsis, PAI-1 gene expression was significantly higher in VAT than in other major organs. VAT PAI-1 gene expression correlated with PAI-1 protein levels in both VAT and plasma. Moreover, VAT and plasma levels of PAI-1 were positively associated with AKI markers, modeling our previous clinical data. Using explant cultures of VAT, we determined that PAI-1 is secreted robustly in response to recombinant transforming growth factor β (TGFβ) and tumor necrosis factor α (TNFα) treatment; however, neutralization was effective only for TNFαindicating that TGFβ is not an endogenous modulator of PAI-1. Within VAT, TNFα was localized to neutrophils and macrophages. PAI-1 protein levels were fourfold higher in stromal vascular fraction (SVF) cells compared with mature adipocytes, and among SVF cells, both immune and nonimmune compartments expressed PAI-1 in a similar fashion. PAI-1 was localized predominantly to macrophages within the immune compartment and preadipocytes and endothelial cells within the nonimmune compartment. Collectively, these results indicate that induction and secretion of PAI-1 from VAT is facilitated by a complex interaction among immune and nonimmune cells. As circulating PAI-1 contributes to AKI in sepsis, understanding PAI-1 regulation in VAT could yield novel strategies for reducing systemic consequences of PAI-1 overproduction.

Elevated postischemic tissue injury and leukocyte-endothelial adhesive interactions in mice with global deficiency in caveolin-2: role of PAI-1.

Ischemia/reperfusion (I/R)-induced rapid inflammation involving activation of leukocyte-endothelial adhesive interactions and leukocyte infiltration into tissues is a major contributor to postischemic tissue injury. However, the molecular mediators involved in this pathological process are not fully known. We have previously reported that caveolin-2 (Cav-2), a protein component of plasma membrane caveolae, regulated leukocyte infiltration in mouse lung carcinoma tumors. The goal of the current study was to examine if Cav-2 plays a role in I/R injury and associated acute leukocyte-mediated inflammation. Using a mouse small intestinal I/R model, we demonstrated that I/R downregulates Cav-2 protein levels in the small bowel. Further study using Cav-2-deficient mice revealed aggravated postischemic tissue injury determined by scoring of villi length in H&E-stained tissue sections, which correlated with increased numbers of MPO-positive tissue-infiltrating leukocytes determined by IHC staining. Intravital microscopic analysis of upstream events relative to leukocyte transmigration and tissue infiltration revealed that leukocyte-endothelial cell adhesive interactions in postcapillary venules, namely leukocyte rolling and adhesion were also enhanced in Cav-2-deficient mice. Mechanistically, Cav-2 deficiency increased plasminogen activator inhibitor-1 (PAI-1) protein levels in the intestinal tissue and a pharmacological inhibition of PAI-1 had overall greater inhibitory effect on both aggravated I/R tissue injury and enhanced leukocyte-endothelial interactions in postcapillary venules in Cav-2-deficient mice. In conclusion, our data suggest that Cav-2 protein alleviates tissue injury in response to I/R by dampening PAI-1 protein levels and thereby reducing leukocyte-endothelial adhesive interactions.NEW & NOTEWORTHY The role of caveolin-2 in regulating ischemia/reperfusion (I/R) tissue injury and the mechanisms underlying its effects are unknown. This study uses caveolin-2-deficient mouse and small intestinal I/R injury models to examine the role of caveolin-2 in the leukocyte-dependent reperfusion injury. We demonstrate for the first time that caveolin-2 plays a protective role from the I/R-induced leukocyte-dependent reperfusion injury by reducing PAI-1 protein levels in intestinal tissue and leukocyte-endothelial adhesive interactions in postcapillary venules.

Berberine dose-dependently inhibits the expression of procoagulant and fibrinolytic inhibitory factors in lipopolysaccharide-induced rat type II alveolar epithelial cells

To observe the effects of berberine on procoagulant and fibrinolytic inhibitory factors produced by rat type II alveolar epithelial cell (AEC II) induced by lipopolysaccharide (LPS). AEC II cells (RLE-6TN cells) were cultured in vitro, and the cells in logarithmic growth phase were collected. The cytotoxicity text of berberine was detected by cell counting kit-8 (CCK-8) to determine the drug concentration range according to inhibition concentration of half cells (IC50). The RLE-6TN cells were divided into five groups, the cells in blank control group were cultured in DMEM; the cells in LPS group were stimulated with 5 mg/L LPS; and the cells in berberine pretreatment groups were pretreated with 20, 50 and 80 μmol/L berberine for 1 hour, and then were co-cultured with 5 mg/L LPS. The cells were collected after LPS induced for 24 hours. The protein and mRNA expression levels of tissue factor (TF), tissue factor pathway inhibitor (TFPI) and plasminogen activator inhibitor-1 (PAI-1) in the cells were detected by Western blotting and real-time fluorescence quantification reverse transcription-polymerase chain reaction (RT-qPCR). The levels of activated protein C (APC), precollagen III peptide (PIIIP), thrombin-antithrombin complex (TAT) and antithrombin III (AT III) in the cell supernatant were measured by enzyme linked immunosorbent assay (ELISA). According to the inhibition rate curve, the IC50 of berberine on RLE-6TN cells was 81.16 μmol/L. Therefore, 20, 50 and 80 μmol/L were selected as the intervention concentration of berberine. Compared with the blank control group, the expression and secretion of procoagulant and fibrinolytic inhibitory factors were abnormal in RLE-6TN cells after LPS induced for 24 hours. The protein and mRNA expression levels of TF and PAI-1 in the LPS group were significantly increased, but the protein and mRNA expression levels of TFPI were significantly decreased. Meanwhile, the levels of APC and AT III in the cell supernatant were significantly decreased, while the levels of PIIIP and TAT were significantly increased. After pretreatment with berberine, the abnormal expression and secretion of procoagulant and fibrinolytic inhibitory factors induced by LPS were corrected in a dose-dependent manner, especially in 80 μmol/L. Compared with the LPS group, the protein and mRNA expression levels of TF and PAI-1 in the berberine 80 μmol/L group were significantly decreased [TF protein (TF/GAPDH): 0.45±0.02 vs. 0.55±0.03, TF mRNA (2-ΔΔCt): 0.39±0.08 vs. 1.48±0.11, PAI-1 protein (PAI-1/GAPDH): 0.37±0.02 vs. 0.64±0.04, PAI-1 mRNA (2-ΔΔCt): 1.14±0.29 vs. 4.18±0.44, all P < 0.01] and those of TFPI were significantly increased [TFPI protein (TFPI/GAPDH): 0.53±0.02 vs. 0.45±0.02, TFPI mRNA (2-ΔΔCt): 0.94±0.08 vs. 0.40±0.05, both P < 0.01]. Meanwhile, the levels of APC and AT III in the cell supernatant were significantly increased [APC (μg/L): 1 358.5±26.0 vs. 994.2±23.1, AT III (μg/L): 118.0±7.4 vs. 84.4±2.7, both P < 0.01], while those of PIIIP and TAT were significantly decreased [PIIIP (μg/L): 11.2±0.4 vs. 18.6±0.9, TAT (ng/L): 222.1±2.8 vs. 287.6±7.0, both P < 0.01]. Berberine could inhibit the LPS-induced expressions of procoagulant and fibrinolytic inhibitory factors in rat AEC II cells and promote the expressions of anticoagulant factors in a dose-dependent manner. Berberine may be a new therapeutic target for alveolar hypercoagulability and fibrinolysis inhibition in acute respiratory distress syndrome (ARDS).

Vepoloxamer Enhances Fibrinolysis of tPA (Tissue-Type Plasminogen Activator) on Acute Ischemic Stroke.

Background and Purpose- Thrombolytic treatment of acute ischemic stroke with tPA (tissue-type plasminogen activator) is hampered by its narrow therapeutic window and potential hemorrhagic complication. Vepoloxamer is a nonionic surfactant that exerts potent hemorheologic and antithrombotic properties in various thrombotic diseases. The current study investigated the effect of vepoloxamer on tPA treatment in a rat model of embolic stroke. Methods- Male Wistar rats subjected to embolic middle cerebral artery occlusion were treated with the combination of vepoloxamer and tPA, vepoloxamer alone, tPA alone, or saline initiated 4 hours after middle cerebral artery occlusion. Results- Monotherapy with tPA did not reduce infarct volume, and adversely potentiated microvascular thrombosis and vascular leakage compared with the saline treatment. Vepoloxamer monotherapy reduced infarct volume by 25% and improved brain perfusion. However, the combination treatment with vepoloxamer and tPA significantly reduced infarct volume by 32% and improved neurological function, without increasing the incidence of gross hemorrhage. Compared with vepoloxamer alone, the combination treatment with vepoloxamer and tPA robustly reduced secondary thrombosis and tPA-augmented microvascular leakage and further improved brain perfusion, which was associated with substantial reductions of serum active PAI-1 (plasminogen activator inhibitor-1) level and tPA-upregulated PAI-1 in the ischemic brain. Mechanistically, exosomes derived from platelets of ischemic rats treated with tPA-augmented cerebral endothelial barrier permeability and elevated protein levels of PAI-1 and TF (tissue factor) in the endothelial cells, whereas exosomes derived from platelets of rats subjected to the combination treatment with vepoloxamer and tPA diminished endothelial permeability augmented by tPA and fibrin and reduced PAI-1 and TF levels in the endothelial cells. Conclusions- The combination treatment with vepoloxamer and tPA exerts potent thrombolytic effects in rats subjected to acute ischemic stroke. Vepoloxamer reduces tPA-aggravated prothrombotic effect of platelet-derived exosomes on cerebral endothelial cells, which may contribute to the therapeutic effect of the combination treatment.

Ashitaba (Angelica Keiskei) Exudate Prevents Increases in Plasminogen Activator Inhibitor-1 Induced by Obesity in Tsumura Suzuki Obese Diabetic Mice

ABSTRACTAngelica keiskei koidzumi (ashitaba) is consumed as a traditional folk medicine and health food in Japan. Ashitaba extract contains abundant flavonoids containing chalcones. Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of tissue plasminogen activator. Excessive amounts of PAI-1 in plasma disrupt the fibrinolytic balance and promote a prothrombotic state with which thrombosis and cardiovascular diseases are associated. In the present study, we investigated the effects of ashitaba yellow exudate (AE) on enhanced PAI-1 levels in Tsumura Suzuki obese diabetic (TSOD) mice. AE significantly decreased food efficiency and plasma PAI-1 in TSOD mice but did not affect lean control Tsumura Suzuki nonobese (TSNO) mice. AE also decreased some parameters in the plasma, such as glucose, insulin, tumor necrosis factor alpha (TNF-α) and gains in body weight, subcutaneous, mesenteric fat weight in TSOD mice but had little effect on these parameters in TSNO mice. Levels of adipose PAI-1 were significantly higher in TSOD than in TSNO mice. Major sources of plasma PAI-1 are thought to be adipose tissue and liver. AE significantly suppressed PAI-1 protein levels in the livers of both TSOD and TSNO mice. These results suggest that AE decreased plasma PAI-1 levels by suppressing both the adipose tissue retention of PAI-1 protein and liver PAI-1 production in TSOD mice. Supplementing the diet with AE might help to prevent thrombotic diseases or alleviate the risk of thrombotic diseases as well as to suppress metabolic state in obese individuals.

Oral citrulline supplementation protects female mice from the development of non-alcoholic fatty liver disease (NAFLD).

Impairments of intestinal barrier function are discussed as risk factors for the development and progression of non-alcoholic fatty liver disease (NAFLD). Studies suggest an association between arginine/citrulline homeostasis and the development of liver damages. Here, the effect of an oral L-citrulline (Cit) supplement on the development of a Western-style diet (WSD)-induced NAFLD was determined in mice. Female 6- to 8-week-old C57BL/6J mice were either pair-fed a liquid Western-style or control diet (C)±2.5g/kg bodyweight Cit for 6weeks (C+Cit or WSD+Cit). Indices of liver damage, glucose metabolism, intestinal barrier function and NO synthesis were measured. While bodyweight gain was similar between groups, markers of glucose metabolism like fasting blood glucose and HOMA index and markers of liver damage like hepatic triglyceride levels, number of neutrophils and plasminogen activator inhibitor-1 protein levels were significantly lower in WSD+Cit-fed mice when compared to WSD-fed mice only. Protein levels of the tight junction proteins occludin and zonula occludens-1 in duodenum were significantly lower in mice fed a WSD when compared to those fed a WSD+Cit (-~70 and -~60%, respectively, P<0.05), whereas portal endotoxin levels, concentration of 3-nitrotyrosine protein adducts in duodenum and toll-like receptor-4 mRNA expression in livers of WSD+Cit-fed mice were markedly lower than in WSD-fed mice (-~43%, P=0.056; -~80 and -~48%, respectively, P<0.05). Our data suggest that the protective effects of supplementing Cit on the development of NAFLD in mice are associated with a decreased translocation of endotoxin into the portal vein.

Plasminogen Activator Inhibitor-1 in depression: Results from Animal and Clinical Studies.

Evidence suggests that plasminogen activator inhibitor-1 (PAI-1) is a stress-related factor, and serum PAI-1 levels are increased in patients with major depressive disorders (MDD). Herein, we analysed PAI-1 protein levels in the brain, cerebrospinal fluid (CSF) and serum of rodents exposed to chronic unpredictable mild stress or treated with escitalopram. In addition, we examined PAI-1 concentrations in serum obtained from 17 drug-free depressed patients before and after escitalopram treatment. We found that PAI-1 expression was increased in area 1 of the cingulate cortex and prelimbic cortex of the medial prefrontal cortex as well as in the hippocampal cornu ammonis 3 and dentate gyrus in stressed rats. A downregulation of PAI-1 following chronic escitalopram treatment was also found. PAI-1 levels were higher in the CSF and serum in stressed rats than in controls, although the difference did not reach statistical significance in the serum. Escitalopram treatment significantly decreased PAI-1 levels in the serum, but not in the CSF. MDD patients had significantly greater serum PAI-1 concentrations than controls. Our results suggest that PAI-1 is implicated in the pathophysiology of depression.

Suppression of plasminogen activator inhibitor-1 by inhaled nitric oxide attenuates the adverse effects of hyperoxia in a rat model of acute lung injury

IntroductionLocally increased expression of plasminogen activator inhibitor-1 (PAI-1) in acute lung injury (ALI) is largely responsible for fibrin deposition in the alveolae and lung microvasculature. In vitro, nitric oxide (NO) effectively suppresses the ischemic induction of PAI-1. We aimed to investigate the effects of inhaled NO on PAI-1 expression in ALI in a rat model with and without hyperoxia. Materials and MethodsHealthy adult rats were primed with lipopolysaccharide (LPS) via an intraperitoneal challenge followed by a second dose of LPS given intratracheally to induce ALI (LPS group), whereas the control groups were given sterile saline. All groups were allocated to subgroups according to gas exposure: NO (20 parts per million, NO), 95% oxygen (O), both (ONO), or room air (A). At 4h, 24h, 48h (after 4h or 24h exposure to the various gases, 24h gas intervention and then observation until 48h), the rat lungs were processed and PAI-1 protein and mRNA expression, histopathological lung injury scores and fibrin deposition were evaluated. ResultsAt 4 and 24h, inhaled NO caused the PAI-1 mRNA levels in the LPS-NO and LPS-ONO subgroups to decrease compared with the untreated LPS subgroups. At 48h, higher PAI-1 mRNA levels than those of the corresponding control subgroup were only observed in the LPS-O subgroup, and these values were lower in the LPS-ONO subgroup than in the LPS-O subgroup. The trends of the PAI-1 protein levels mirrored those of PAI-1 mRNA. At 48h, PAI-1 protein levels in the LPS-NO and LPS-ONO subgroups were decreased compared with those in the untreated LPS subgroups. The histopathological lung injury scores and fibrin deposition in LPS subgroups that inhaled NO showed a decreasing trend compared with the untreated LPS subgroups. ConclusionsInhaled NO can suppress elevated PAI-1 expression in rats with ALI induced by endotoxin. Although exposure to high-concentration oxygen prolongs the duration of PAI-1 mRNA overexpression in ALI, inhaled NO can reduce this effect and alleviate both fibrin deposition and lung injury.

A small molecule PAI-1 functional inhibitor attenuates neointimal hyperplasia and vascular smooth muscle cell survival by promoting PAI-1 cleavage

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of urokinase-and tissue-type plasminogen activators (uPA and tPA), is an injury-response gene implicated in the development of tissue fibrosis and cardiovascular disease. PAI-1 mRNA and protein levels were elevated in the balloon catheter-injured carotid and in the vascular smooth muscle cell (VSMC)-enriched neointima of ligated arteries. PAI-1/uPA complex formation and PAI-1 antiproteolytic activity can be inhibited, via proteolytic cleavage, by the small molecule antagonist tiplaxtinin which effectively increased the VSMC apoptotic index in vitro and attenuated carotid artery neointimal formation in vivo. In contrast to the active full-length serine protease inhibitor (SERPIN), elastase-cleaved PAI-1 (similar to tiplaxtinin) also promoted VSMC apoptosis in vitro and similarly reduced neointimal formation in vivo. The mechanism through which cleaved PAI-1 (CL-PAI-1) stimulates apoptosis appears to involve the TNF-α family member TWEAK (TNF-α weak inducer of apoptosis) and it's cognate receptor, fibroblast growth factor (FGF)-inducible 14 (FN14). CL-PAI-1 sensitizes cells to TWEAK-stimulated apoptosis while full-length PAI-1 did not, presumably due to its ability to down-regulate FN14 in a low density lipoprotein receptor-related protein 1 (LRP1)-dependent mechanism. It appears that prolonged exposure of VSMCs to CL-PAI-1 induces apoptosis by augmenting TWEAK/FN14 pro-apoptotic signaling. This work identifies a critical, anti-stenotic, role for a functionally-inactive (at least with regard to its protease inhibitory function) cleaved SERPIN. Therapies that promote the conversion of full-length to cleaved PAI-1 may have translational implications.

The -844 G>A PAI-1 polymorphism is associated with acute coronary syndrome in Mexican population.

Background. Acute coronary syndrome (ACS) has an important impact in public health with high morbidity and mortality. Prothrombotic and proinflammatory states are involved in the pathogenesis of the disease. Plasminogen activator inhibitor-1 (PAI-1) is the major inhibitor of the fibrinolysis and also is part of immune response. The -844 G>A gene polymorphism is related to increased PAI-1 protein levels. The aim of the study is to evaluate the association of -844 G>A PAI-1 polymorphism with ACS. Methods. A total of 646 individuals were recruited from Western Mexico: 350 unrelated healthy subjects and 296 patients with diagnosis of ACS. Results. The most important risk factor in our population was hypertension, followed by smoking. The genetic distribution showed an association of the A allele (OR = 1.27, P = 0.04) and AA genotype (OR = 1.86, P = 0.02) with ACS. The recessive model displayed similar results (OR = 1.76, P = 0.02). As additional finding, we observed significant differences in the genetic distribution of ACS dyslipidemic patients (OR = 1.99, P = 0.04). The A allele and AA genotype of -844 polymorphism of PAI-1 gene are risk factors for ACS. The AA genotype might be associated with the development of dyslipidemia in ACS patients.

Plasminogen activator inhibitor-1 regulates LPS-induced TLR4/MD-2 pathway activation and inflammation in alveolar macrophages.

Toll-like receptor 4 (TLR4) and myeloid differentiation protein 2 (MD-2) are the main lipopolysaccharide (LPS) binding receptors that respond to inflammatory stimuli and mediate NF-kappa B (NF-κB) signaling pathway in macrophages. We have previously shown that plasminogen activator inhibitor-1 (PAI-1) deletion increased lung injury induced by intratracheal instillation of LPS through downregulation of TLR4 negative regulators. However, the mechanisms by which PAI-1 regulates lung inflammation are largely unknown. The aim of this study is to assess the relationship between PAI-1 and TLR4 signaling pathways in LPS-induced NR8383 cells inflammatory reaction. The results showed that the levels of PAI-1, TNF-α, and IL-1β protein were increased remarkably in NR8383 cell supernatants after LPS stimulation. PAI-1 gene knockdown reduced TNF-α and IL-1β levels in cell supernatants and inhibited the NF-κB p65 protein expression in NR8383 cells. The upregulated mRNA and protein expressions of TLR4, MD-2, and myeloid differentiation protein (MyD88) induced by LPS were attenuated after PAI-1 gene knockdown. Conversely, overexpression of PAI-1 in NR8383 cells not only resulted in additional mRNA and protein production of PAI-1, TLR4, MD-2, and MyD88, it also aggravated the inflammatory response induced by LPS. In conclusion, PAI-1 contributes to the regulation of LPS-induced inflammatory response in NR8383 cells, likely by affecting the TLR4-MD-2/NF-κB signaling transduction pathway.

Levels of microRNA-181b and plasminogen activator inhibitor-1 are associated with hypertensive disorders complicating pregnancy.

The aim of the present study was to explore the association between the expression of microRNA (miRNA)-181b and plasminogen activator inhibitor-1 (PAI-1) in the placental tissue of pregnant females with a hypertensive disorder complicating pregnancy (HDCP). Placental tissue samples were obtained from 48 patients with HDCP and 40 females with a normal pregnancy. The levels of miRNA-181b and PAI-1 mRNA were determined by the reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expression of PAI-1 protein was analyzed by western blotting. Vascular smooth muscle cells (VSMCs) were transfected with the pEGP-miRNA-181b plasmid using Lipofectamine® 2000. Transfection efficiency was confirmed by immunohistochemical analysis. The levels of miRNA-181b in the placental tissue of patients with HDCP were lower than those in the control group, whereas the levels of PAI-1 mRNA in the placental tissue of patients with HDCP were higher than those in the control group. The expression of the PAI-1 protein in the HDCP group was higher than that in the control group. Following transfection of VSMCs with plasmid pGCMV/EGFP/miRNA-181b, the levels of PAI-1 mRNA were reduced while the levels of miRNA-181 were upregulated. Furthermore, the expression levels of PAI-1 protein were lower than those in the control group. The levels of miRNA-181b and PAI-1 mRNA were strongly associated with HDCP. Thus, miRNA-181b may play an important role in the regulation of PAI-1. PAI-1 and miRNA-181b may be novel biomarkers to be used in HDCP therapy.

Mechanical ventilation augments bleomycin-induced epithelial–mesenchymal transition through the Src pathway

Mechanical ventilation used in patients with acute respiratory distress syndrome (ARDS) can damage pulmonary epithelial cells by producing inflammatory cytokines and depositing excess collagen. Src participates in plasminogen activator inhibitor-1 (PAI-1) and transforming growth factor-β1(TGF-β1) production during the fibroproliferative phase of ARDS, which involves a process of epithelial–mesenchymal transition (EMT). The mechanisms regulating interactions between mechanical ventilation and EMT are unclear. We hypothesized that EMT induced by high-tidal volume (VT) mechanical stretch-augmented lung inflammation occurs through upregulation of the Src pathway. Five days after administering bleomycin to simulate acute lung injury (ALI), male C57BL/6 mice, either wild-type or Src-deficient, aged 3 months, weighing between 25 and 30 g, were exposed to low-VT (6 ml/kg) or high-VT (30 ml/kg) mechanical ventilation with room air for 1–5 h. Nonventilated mice were used as control subjects. We observed that high-VT mechanical ventilation increased microvascular permeability, PAI-1 and TGF-β1 protein levels, Masson's trichrome staining, extracellular collagen levels, collagen gene expression, fibroblast accumulation, positive staining of α-smooth muscle actin and type I collagen, activation of Src signaling and epithelial apoptotic cell death in wild-type mice (P<0.05). Decreased staining of the epithelial marker, Zonula occludents-1, was also observed. Mechanical stretch-augmented EMT and epithelial apoptosis were attenuated in Src-deficient mice and pharmacological inhibition of Src activity by PP2 (P<0.05). Our data suggest that high-VT mechanical ventilation-augmented EMT after bleomycin-induced ALI partially depends on the Src pathway.

The urea decomposition product cyanate promotes endothelial dysfunction.

The dramatic cardiovascular mortality of patients with chronic kidney disease is attributable in a significant proportion to endothelial dysfunction. Cyanate, a reactive species in equilibrium with urea, is formed in excess in chronic kidney disease. Cyanate is thought to have a causal role in promoting cardiovascular disease, but the underlying mechanisms remain unclear. Immunohistochemical analysis performed in the present study revealed that carbamylated epitopes associate mainly with endothelial cells in human atherosclerotic lesions. Cyanate treatment of human coronary artery endothelial cells reduced expression of endothelial nitric oxide synthase, and increased tissue factor and plasminogen activator inhibitor-1 expression. In mice, administration of cyanate, promoting protein carbamylation at levels observed in uremic patients, attenuated arterial vasorelaxation of aortic rings in response to acetylcholine without affecting the sodium nitroprusside-induced relaxation. Total endothelial nitric oxide synthase and nitric oxide production were significantly reduced in aortic tissue of cyanate-treated mice. This coincided with a marked increase of tissue factor and plasminogen activator inhibitor-1 protein levels in aortas of cyanate-treated mice. Thus, cyanate compromises endothelial functionality in vitro and in vivo. This may contribute to the dramatic cardiovascular risk of patients suffering from chronic kidney disease.

Abstract 280: Increased Metabolite Levels of Glycolysis and Pentose Phosphate Pathway in Rabbit Atherosclerotic Arteries and Hypoxic Macrophage

Aims: Inflammation and possibly hypoxia largely affect glucose utilization in atherosclerotic arteries, which could alter many metabolic systems. However, metabolic changes in atherosclerotic plaques remain unknown. The present study aims to identify changes in metabolic systems relative to glucose uptake and hypoxia in rabbit atherosclerotic arteries and cultured macrophages. Methods: Macrophage-rich or smooth muscle cell (SMC)-rich neointima was created by balloon injury in the iliac-femoral arteries of rabbits fed with a 0.5% cholesterol diet or a conventional diet. THP-1 macrophages stimulated with lipopolysaccharides (LPS) and interferon-γ (INFγ) were cultured under normoxic and hypoxic conditions. We evaluated comprehensive arterial and macrophage metabolism by performing metabolomic analyses using capillary electrophoresis-time of flight mass spectrometry. We evaluated glucose uptake and its relationship to vascular hypoxia using 18F-fluorodeoxyglucose (18F-FDG) and pimonidazole, a marker of hypoxia. Results: The levels of many metabolites increased in the iliac-femoral arteries with macrophage-rich neointima, compared with those that were not injured and those with SMC-rich neointima (glycolysis, 4 of 9; pentose phosphate pathway, 4 of 6; tricarboxylic acid cycle, 4 of 6; nucleotides, 10 of 20). The uptake of 18F-FDG in arterial walls measured by autoradiography positively correlated with macrophage- and pimonidazole-immunopositive areas (r = 0.76, and r = 0.59 respectively; n = 69 for both; p &lt; 0.0001). Pimonidazole immunoreactivity was closely localized with the nuclear translocation of hypoxia inducible factor-1α and hexokinase II expression in macrophage-rich neointima. The levels of glycolytic (8 of 8) and pentose phosphate pathway (4 of 6) metabolites increased in LPS and INFγ stimulated macrophages under hypoxic but not normoxic condition. Plasminogen activator inhibitor-1 protein levels in the supernatant were closely associated with metabolic pathways in the macrophages. Conclusion: Infiltrative macrophages in atherosclerotic arteries might affect metabolic systems, and hypoxia but not classical activation might augment glycolytic and pentose phosphate pathways in macrophages.

Increased Metabolite Levels of Glycolysis and Pentose Phosphate Pathway in Rabbit Atherosclerotic Arteries and Hypoxic Macrophage

AimsInflammation and possibly hypoxia largely affect glucose utilization in atherosclerotic arteries, which could alter many metabolic systems. However, metabolic changes in atherosclerotic plaques remain unknown. The present study aims to identify changes in metabolic systems relative to glucose uptake and hypoxia in rabbit atherosclerotic arteries and cultured macrophages.MethodsMacrophage-rich or smooth muscle cell (SMC)-rich neointima was created by balloon injury in the iliac-femoral arteries of rabbits fed with a 0.5% cholesterol diet or a conventional diet. THP-1 macrophages stimulated with lipopolysaccharides (LPS) and interferon-γ (INFγ) were cultured under normoxic and hypoxic conditions. We evaluated comprehensive arterial and macrophage metabolism by performing metabolomic analyses using capillary electrophoresis-time of flight mass spectrometry. We evaluated glucose uptake and its relationship to vascular hypoxia using 18F-fluorodeoxyglucose (18F-FDG) and pimonidazole, a marker of hypoxia.ResultsThe levels of many metabolites increased in the iliac-femoral arteries with macrophage-rich neointima, compared with those that were not injured and those with SMC-rich neointima (glycolysis, 4 of 9; pentose phosphate pathway, 4 of 6; tricarboxylic acid cycle, 4 of 6; nucleotides, 10 of 20). The uptake of 18F-FDG in arterial walls measured by autoradiography positively correlated with macrophage- and pimonidazole-immunopositive areas (r = 0.76, and r = 0.59 respectively; n = 69 for both; p<0.0001). Pimonidazole immunoreactivity was closely localized with the nuclear translocation of hypoxia inducible factor-1α and hexokinase II expression in macrophage-rich neointima. The levels of glycolytic (8 of 8) and pentose phosphate pathway (4 of 6) metabolites increased in LPS and INFγ stimulated macrophages under hypoxic but not normoxic condition. Plasminogen activator inhibitor-1 protein levels in the supernatant were closely associated with metabolic pathways in the macrophages.ConclusionInfiltrative macrophages in atherosclerotic arteries might affect metabolic systems, and hypoxia but not classical activation might augment glycolytic and pentose phosphate pathways in macrophages.

Expression of toll‐like receptors 1–5 but not TLR 6–10 is elevated in livers of patients with non‐alcoholic fatty liver disease

Animal models of non-alcoholic fatty liver disease (NAFLD) suggest that an increased translocation of bacterial endotoxins, leading to an activation of toll-like receptor-dependent signalling cascades (TLRs) and increased formation of reactive oxygen species, may add to development of insulin resistance and induction of plasminogen activator inhibitor-1 (PAI-1) in the liver. If similar mechanisms are also involved in the development of NAFLD in humans remains to be determined. Toll-like receptor (1-10), myeloid differentiation primary response gene (MyD88), interferon regulatory transcription factor 3 (IRF-3) and insulin receptor substrate 1 (IRS-1) mRNA expression was determined in liver samples of 11 patients with NAFLD and 11 controls. Hepatic PA1-1 and 4-hydroxynonenal protein adducts (4-HNE) levels were determined by immunohistochemistry. Hepatic TLR 1-5 mRNAs expression was significantly higher in livers of NAFLD patients than in controls, whereas expression of TLR 6-10 mRNAs did not differ between groups. Expression of MyD88 but not IRF-3 was also significantly higher in livers of NAFLD patients than in controls. These alterations were associated with significantly higher levels of 4-HNE and PAI-1 protein levels in livers of NAFLD patients than in controls, whereas IRS-1 mRNA expression was ~80% lower in livers of NAFLD patients than in controls. Taken together, these findings add further weight to the hypothesis that alterations at the level of intestine and intestinal barrier function may be critical in the development of NAFLD in humans.

Plasminogen activator inhibitor-1 regulates LPS induced inflammation in rat macrophages through autophagy activation.

Background. The mechanisms by which plasminogen activator inhibitor-1 (PAI-1) regulates inflammation, especially in acute respiratory distress syndrome (ARDS), are largely unknown. Objective. To assess the relationship between PAI-1 and autophagy in inflammatory reactions induced by LPS in rat NR8383 cells. Methods. ELISA was used to assess the amounts of TNF-α, IL-1β, and PAI-1 in cell culture supernatants; TLR4, MyD88, PAI-1, LC3, Beclin1, and mTOR protein and mRNA levels were determined by western blot and quantitative RT-PCR, respectively; western blot was used to determine NF-κB protein levels. To further evaluate the role of PAI-1, the PAI-1 gene was downregulated and overexpressed using the siRNA transfection technology and the pCDH-PAI-1, respectively. Finally, the GFP Positive Expression Rate Method was used to determine the rate of GFP-LC3 positive NR8383 cells. Results. In LPS-induced NR8383 cells, TNF-α, IL-1β, and PAI-1 expression levels increased remarkably. Upon PAI-1 knockdown, TNF-α, IL-1β, PAI-1, TLR4, MyD88, NF-κB, LC3, and Beclin1 levels were decreased, while mTOR increased. Conversely, overexpression of PAI-1 resulted in increased amounts of TNF-α, IL-1β, PAI-1, TLR4, MyD88, NF-κB, LC3, and Beclin1. However, no significant change was observed in mTOR expression. Conclusions. In NR8383 cells, PAI-1 contributes in the regulation of LPS-induced inflammation, likely by promoting autophagy.