Plasminogen Activator Inhibitor-1 mRNA Levels Research Articles

Rho-Kinase Mediates Hyperglycemia-Induced Plasminogen Activator Inhibitor-1 Expression in Vascular Endothelial Cells

Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are associated with myocardial infarction and stroke, especially in patients with diabetes. The induction of PAI-1 expression by hyperglycemia involves oxidative stress and protein kinase C (PKC). However, the mechanism by which hyperglycemia increases PAI-1 expression is unknown. Compared with normoglycemia, exposure of human endothelial cells to hyperglycemia, but not mannitol, increased Rho-kinase activity in a time- and concentration-dependent manner. This increase was inhibited by a PKC inhibitor, GF109203X, and antioxidants N-acetylcysteine (NAC) and reduced form of glutathione (GSH). This correlated with inhibition of hyperglycemia-induced PAI-1 expression by GF109203X, NAC, and GSH. Hyperglycemia-increased PAI-1 mRNA and protein levels were inhibited by Rho-kinase inhibitors hydroxyfasudil and Y27632 and by a dominant-negative mutant of Rho-kinase. The mechanism for this inhibition occurs at the level of gene transcription because Rho-kinase inhibitors repress hyperglycemia-stimulated PAI-1 promoter activity without affecting mRNA stability. Hyperglycemia failed to stimulate Rho-kinase activity and PAI-1 expression in heterozygous ROCK I-knockout murine endothelial cells. Hyperglycemia stimulates Rho-kinase activity via PKC- and oxidative stress-dependent pathways, leading to increased PAI-1 gene transcription. These results suggest that inhibition of ROCK I may be a novel therapeutic target for preventing thromboembolic complications of diabetes and cardiovascular disease.

Chronic Losartan Therapy in OLETF Rats Has a Differential Effect on Adipose and Vascular PAM

Angiotensinogen (AGT) and plasminogen activator inhibitor-1 (PAI-1) are expressed in both vascular and adipose tissues. Angiotensin II (AG II) has an adipogenic effect and increases PAI-1 expression. To evaluate the chronic effects of AG II type 1 receptor (AT1R) antagonism on adipose mass and PAI-1 expression in vascular and adipose tissues, losartan (30 mg/kg/day) was administered to Otsuka Long Evans Tokushima Fatty (OLETF) rats, a model of type 2 diabetes, for 20 weeks. Adipose mass and regional fat distribution in the abdomen did not change after chronic AT1R antagonism in OLETF rats. AGT and PAI-1 mRNA expressions in adipose tissue of OLETF rats were significantly increased compared with Long-Evans Tokushima Otsuka (LETO) rats, the normal control. Chronic losartan therapy further increased the level of adipose AGT in OLETF rats, but did not affect the level of adipose PAI-1 mRNA. In contrast, aortic PAI-1 protein expression, assessed by immunofluorescent staining, was attenuated by chronic losartan therapy. Our results have three implications. First, chronic AT1R antagonism with losartan increases AGT expression in adipose tissue; increased AGT may sustain PAI-1 expression in adipose tissue despite the AT1R blockade. Second, chronic losartan therapy does not affect adipose mass. Third, chronic AT1R antagonism with losartan has a differential effect on vascular and adipose PAI-1 expression.

Aldosterone increases plasminogen activator inhibitor-1 synthesis in rat cardiomyocytes

Plasminogen activator inhibitor-1 (PAI-1) is an anti-thrombolytic factor that also promotes tissue fibrosis. Under certain conditions, exposure to aldosterone can result in cardiac fibrosis by an unknown mechanism. In the current study, we tested the hypothesis that PAI-1 is a mediator of aldosterone's fibrotic effects. Aldosterone increased levels of PAI-1 mRNA and protein in the H9c2 rat cardiac cell line, responses that could be blocked by the mineralocorticoid receptor (MR) antagonist spironolactone. Confocal microscopy confirmed an effect of aldosterone to increase PAI-1 expression with reversal by spironolactone. Aldosterone also increased PAI-1 expression in neonatal rat cardiomyocytes, which was again blocked by spironolactone. In the neonatal cardiomyocytes (but not the H9c2 cells), anti-transforming growth factor-β1 antibody inhibited the PAI-1 response to aldosterone. In summary, aldosterone directly increased PAI-1 expression in two different cardiac muscle cell types, an effect that was dependent on MR. In the neonatal cells, there appeared to be a requirement for transforming growth factor-β1.

Inflammatory Cytokines Interleukin-6 and Oncostatin M Induce Plasminogen Activator Inhibitor-1 in Human Adipose Tissue

Adipose tissue is a prominent source of plasminogen activator inhibitor-1 (PAI-1), the primary physiological inhibitor of plasminogen activation. Increased PAI-1 expression acts as a cardiovascular risk factor, and plasma levels of PAI-1 strongly correlate with body mass index (BMI). Elevated serum levels of interleukin-6 (IL-6), an inflammatory cytokine and a member of the glycoprotein 130 (gp130) ligand family, are found in obese patients and might indicate low-grade systemic inflammation. Another gp130 ligand, oncostatin M (OSM), upregulates PAI-1 in cardiac myocytes, astrocytes, and endothelial cells. We used tissue explants and primary cultures of preadipocytes and adipocytes from human subcutaneous and visceral adipose tissue to investigate whether IL-6 and OSM affect PAI-1 expression in fat. Human subcutaneous and visceral adipose tissue responded to treatment with IL-6 and OSM with a significant increase in PAI-1 production. Human preadipocytes were isolated from subcutaneous and visceral adipose tissue. Adipocyte differentiation was induced by hormone supplementation. All cell types expressed receptors for IL-6 and OSM and produced up to 12-fold increased levels of PAI-1 protein and up to 9-fold increased levels of PAI-1 mRNA on stimulation with IL-6 and OSM. AG-490, a janus kinase/signal transducer and activator of transcription inhibitor, abolished the OSM-dependent PAI-1 induction almost completely. We have for the first time established a link between the gp130 ligands, the proinflammatory mediators IL-6 and OSM, and the expression of PAI-1 in human adipose tissue. Thus, we speculate that IL-6 and OSM, by upregulating PAI-1 in adipose tissue, can contribute to the increased cardiovascular risk of obese patients.

Enhancement of PAI-1 mRNA in cardiovascular cells after kainate injection is mediated through the sympathetic nervous system

Plasminogen activator inhibitor-1 (PAI-1) is a major physiological regulator of the fibrinolytic system and is thought to promote vascular diseases. Recently, we have reported that PAI-1 gene expression was markedly enhanced locally in cardiovascular cells immediately after injecting (i.p.) mice with kainate, an analog of glutamate, which is the principal excitatory neurotransmitter in the central nervous system. Here we investigated whether the induction of PAI-1 mRNA by kainate could be mediated through sympathetic versus parasympathetic efferent neurons. To this end, we used a group of drugs known to interfere with the aforementioned pathways. PAI-1 gene expression was monitored in the heart via in situ hybridization using 35S-labeled PAI-1-specific riboprobes. We have found that the elevation of PAI-1 mRNA levels, as detected 3 h after systemic administration of kainate, was reduced by mecamylamine, guanethidine and phentolamine, but not by propranolol or atropine. In addition, the adrenergic agonists phenylephrine and adrenaline themselves, but not clonidine, induced PAI-1 with a spatial distribution similar to that of kainate (i.e. in coronary arteries throughout the heart, and in cardiocytes in the left ventricular and atrial myocardium). Collectively, these results show that kainate activated the PAI-1 gene in cardiovascular cells primarily through the sympathetic nervous system (SNS), via the α1-adrenergic receptor. Hence, the results suggest that PAI-1 is likely to be increased during enhanced sympathetic efferent neuronal activity, such as occurring in heart failure or cardiac hypertrophy. The results also reinforce the previously reported linkage of PAI-1 to physiological stress. Furthermore, to our knowledge, this is the first demonstration that glutamate can enhance gene expression in a peripheral tissue. Thus, these findings raise the possibility that glutamate, acting via the SNS, can affect cardiovascular homeostasis and pathology by modulating gene expression in cardiac myocytes and vascular cells.

Endothelin‐mediated remodeling in aortas of diabetic rats

Smooth muscle cells proliferation and extracellular matrix (ECM) protein deposition are key features of diabetic macroangiopathy. In the present study, we have studied the role of endothelin(A) (ET(A)) receptor, the predominant receptor on smooth muscle cells, in diabetes-induced vascular hypertrophy and remodeling. Streptozotocin-induced diabetic rats were administrated a selective ET(A) receptor antagonist, TBC3214, for 26 weeks. Following treatment, aortas were harvested and subjected to gene expression and morphometric analyses. We quantified fibronectin (FN) and plasminogen activator inhibitor-1 (PAI-1) expression as indicators of increased ECM protein synthesis. ET-1, ET-3, transforming growth factor-beta1 (TGF-beta1) and angiotensinogen mRNA levels were measured to elucidate genes involved in FN expression. We have investigated an embryonic splice variant of FN, oncofetal FN, and nonmuscle myosin heavy chain (SMemb) as vascular remodeling indicators. Our results show that diabetes leads to upregulation of FN, PAI-1, ET-1, ET-3, TGF-beta1 and angiotensinogen mRNA levels in association with increased medial thickness. Immunohistochemical analyses revealed concurrent protein level changes. Diabetes also upregulated oncofetal FN and SMemb mRNA levels. Treatment with TBC3214 attenuated the mRNA levels of several genes and prevented increased medial thickness. These results indicate that diabetes-induced vascular hypertrophy and remodeling is associated with reexpression of embryonic forms of FN and myosin heavy chain. Such changes are ET-dependent and may be mediated via TGF-beta1 and angiotensin.

Hormonal Regulation of Leptin, Resistin, and Plasminogen Activator Inhibitor-1 Gene Expression in 3T3-L1 Adipocytes

Leptin, resisitn and PAI-1 (plasminogen activator inhibitor-1) are synthesized and secreted by rodent fat cells and recently postulated to be an important link to obesity. This study was conducted to characterize the hormonal regulation of leptin, resistin, and PAI-1 gene expression in the 3T3-L1 adipocytes. The cells were treated with 0.5 μM insulin, 1 μM dexamethasone (Dex), or 0.05 μM triiodothyronine (T3) for 72 hours. The mRNA levels of each peptide were measured by semi-quantitative RT-PCR. The mRNA level of the leptin-producing ob gene was significantly increased by insulin, Dex, and T3 by 3.2-, 3.1- and 2.7-fold, respectively, compared to the control (p＜0.05). The level of resistin mRNA was increased by insulin, Dex, and T3 by 2.7-, 2.5- and 2-fold, respectively, compared to the control (p＜0.05). Likewise, the level of PAI-1 mRNA was significantly increased by insulin, Dex, and T3 compared to the control (p＜0.05). Taken together, our results suggest that insulin, Dex, and T3 may regulate the gene expression of leptin, resistin, and PAI-1 in 3T3-L1 adipocytes.

Enhanced plasminogen activator inhibitor-1 expression in transgenic mice with hepatocyte-specific overexpression of superoxide dismutase or glutathione peroxidase.

In this study, we developed a double-transgenic mouse model allowing hepatocyte-specific and regulated expression of the redox-modifying enzymes copper/zinc superoxide dismutase (SOD) and glutathione peroxidase (GPX) by using a tetracycline-regulatable gene expression system. Within this system, the SOD and GPX level can be regulated deliberately by addition or removal of doxycycline hydrochloride to the drinking water. As reactive oxygen species (ROS) have been implicated in a number of pathological conditions, such as atherosclerosis, thrombosis, or liver fibrosis, processes that are also frequently associated with enhanced levels of plasminogen activator inhibitor-1 (PAI-1), it was the aim of the present study to investigate the influence of SOD and GPX overexpression on the regulation of PAI-1. PAI-1 mRNA and protein levels in tetracycline transactivator-dependent SOD-overexpressing double-transgenic mice reached values 2.5- to threefold above the normal mRNA level. By applying doxycycline, a deinduction of the PAI-1 levels was observed. By using the same protocol, PAI-1 mRNA and protein levels were enhanced in GPX double-transgenic mice, and again this response was blunted by the addition of doxycycline. These studies provide some new information regarding the role of ROS within the proteolytic processes in hepatocytes that require PAI-1.

PAI-1 GENE EXPRESSION IS INDUCED BY FLUID SHEAR STRESS IN RAT HEPATOCYTE

A186 Aims: Liver sinusoidal cells are always exposed to mechanical stress arising from sinusoidal blood flow. In the remnant liver after hepatectomy or non occluded lobe in the portal branch embolization or graft liver in liver transplantation, it is possible that increasing shear stress effects hepatocyte directory and modulate liver regeneration or liver damage. Some investigators have shown its possibility but there are few reports about shear stress and gene expression of hepatocyte. Plasminogen activator inhibitor-1 (PAI-1) is known to be induced transiently in the early phase of liver regeneration but its mechanism and function are still unclear. We loaded shear stress to the cultured hepatocyte and studied PAI-1 gene expression. Methods: 1. Rat hepatocyte were cultured on glass plate and set in the parallel chamber. Fluid shear stress was loaded to the hepatocyte. PAI-1 mRNA expression was analyzed by the real time PCR and compared with static control. Luciferase assay and nuclear run-on asasy were performed to study their transcriptional activity. 2. Wistar rat weighed 450g was used to confirm hemodynamic change. Portal vein branch responsible for about 70% of liver was ligated and portal vein pressure was measured by direct cannulation and sinusoidal blood velocity, flow rate and vascular area were analyzed in non ligated lobe by use of laser doppler flow meter. Then PAI-1 mRNA expression in the non ligated liver tissue was also studied. 3. Plasma PAI-1 level in the recipients of living donor liver transplantation were measured before and after reperfusion of the graft liver. Results: After loading shear stress (10dynes/cm2) for six hours PAI-1 mRNA expression was increased transiently to 6.7 fold of static control. Compared with normal medium and that added 5% dextran, PAI-1 mRNA expression was induced more in the latter at any flow rate. Transcriptional activity was also increased. Portal pressure was increased after portal branch ligation and sinusoidal blood velocity and flow rate were increased 40% significantly. PAI-1 mRNA level in non ligated liver tissue was risen up to 40 times at three hours after ligation. Plasma PAI-1 level after blood reperfusion was higher than before perfusion. Conclusions: PAI-1 mRNA expression was induced by fluid shear stress in rat hepatocyte. It is suggested that PAI-1 gene expression in the remnant liver after hepatectomy or graft liver in liver transplantation is induced by increased shear stress arisen from sinusoidal blood flow.

Effect of Tisseel® on expression of tissue plasminogen activator and plasminogen activator inhibitor-1

Objective To examine the effect of fibrin sealant on mRNA expression of factors regulating plasminogen activator activity in human peritoneal cells. Plasminogen activator activity is thought to play a pivotal role in degradation of the proteinaceous mass that develops after surgical procedures. Reduction of plasminogen activator activity, as occurs with tissue trauma, results in increased postoperative adhesion development. Design Tissue culture for 6, 12, 24, and 48 hours. Setting University research laboratory. Patients Source of mesothelial cells with fibroblasts. Intervention(s) Measurement of mRNA expression of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1). Main outcome measure(s) Multiplex reverse transcriptase/polymerase chain reaction (RT/PCR) was used to determine relative change in t-PA and PAI-1 mRNA levels under six conditions: [1] fibrin sealant (Tisseel®); [2] fibrin sealant (Tisseel®) two components diluted 1:2; [3] fibrin sealant (Tisseel®) sealer protein component reconstituted without aprotinin (a protease inhibitor); [4] fibrin sealant (Tisseel®) sealer protein component reconstituted without aprotinin, both components diluted 1:2; [5] fibrin sealant (Tisseel®) components diluted to physiologic concentrations; and [6] control (culture media). Results The mRNA levels of t-PA and PAI-1 by human peritoneal cells were unchanged during 48 hours. In mesothelial cells, the addition of the compositions increased t-PA mRNA levels. A selective increase was observed in the normal peritoneal fibroblasts at the later time points; similar increases were identified in adhesion fibroblast cultures. In mesothelial cells, the more concentrated compositions generally increased PAI-1 mRNA above control levels, whereas in normal peritoneal fibroblasts PAI-1 levels generally remained unchanged. In contrast, in adhesion fibroblasts, PAI-1 levels decreased over time with treatment. Conclusion(s) Fibrin sealant, in the presence and absence of aprotinin, increases both t-PA and PAI-1 expression by human peritoneal cells; changes not seen with physiologic concentrations of fibrin sealant. These observations suggest that in addition to its ability to help achieve hemostasis, fibrin sealant affects the healing process by altering components of the plasminogen activator system, which may be of benefit in the reduction of postoperative adhesions.

The PAI-1 gene as a direct target of endothelial PAS domain protein-1 in adenocarcinoma A549 cells.

Endothelial PAS domain protein-1 (EPAS1) regulates transcription of the genes encoding erythropoietin and vascular endothelial growth factor, which are important for maintaining oxygen homeostasis. We have previously shown that plasminogen activator inhibitor-1 (PAI-1) gene expression is induced by hypoxia. In this study, we sought to determine whether PAI-1 gene expression is directly regulated by EPAS1 in cancer cells because activities of proteases and their inhibitors are tightly regulated for tumor invasion. Hypoxia increased the PAI-1 mRNA levels in human adenocarcinoma A549 cells. Overexpression of EPAS1 significantly increased the PAI-1 mRNA and protein levels. Transient transfection assays revealed that EPAS1 increased PAI-1 gene transcription through a sequence containing 5'-CACGTACA-3' located at -194 (we refer to it as site HREPAI-1) and GT-box located at -78. Electrophoretic gel mobility shift assays revealed that HREPAI-1 serves as a binding site for EPAS1, and Sp1 constitutively binds to GT-box. In conclusion, PAI-1 expression is induced by EPAS1 through HREPAI-1 and through an Sp1-binding site. These results indicate that the PAI-1 gene is a direct target of EPAS1 and suggest the role of EPAS1 and Sp1 in the hypoxic response of cancer cells.

Plasminogen activator inhibitor-1 deficiency enhances flow-induced smooth muscle cell migration

Introduction: We determined the role of smooth muscle cell (SMC)-derived plasminogen activator inhibitor-1 (PAI-1) in the flow-induced SMC migratory response. Materials and methods: Wild type (wt) or PAI-1 knockout SMC were cultured in the absence or presence of endothelial cells (EC) under static or pulsatile flow conditions in a perfused culture system. SMC migration was then assessed by Transwell assay. Results: Pulsatile flow significantly increased SMC PAI-1 mRNA and protein levels, ∼4- and 3-fold respectively ( n=4, p<0.05). In the absence, but not in the presence of EC, pulsatile flow significantly increased (∼2.4-fold) the migration of wt SMC when compared to wt SMC cultured under static conditions. PAI-1 −/−SMC migration was significantly increased under flow conditions as compared to wild-type controls (334±22% vs. 237±11%, n=6, p<0.05). This flow-induced migration was significantly attenuated, but not completely inhibited, when PAI-1 −/−SMC were cultured in the presence of EC (147±13%, n=6, p<0.05). The flow-induced PAI-1 −/−SMC migratory response was partially inhibited by an anti-urokinase plasminogen activator (uPA) antibody (#1189), and completely inhibited by both 1189 and the matrix metalloproteinase (MMP) inhibitor BB3103. In parallel PAI-1 −/−SMC cells, there was a greater flow-induced increase in proMMP-2 activity as compared to wild-type control cells. Moreover, under both static and flow conditions, tissue inhibitors of matrix metalloproteinases (TIMP)-2 activity was reduced in these PAI-1-deficient cells as compared to wild-type controls. Conclusions: These results suggest that SMC PAI-1 plays a role in limiting flow-induced SMC migration and thus may be an important mechanism for controlling the process of vascular remodelling.

Minimally oxidized low-density lipoprotein regulates hemostasis factors of brain capillary endothelial cells

Minimally oxidized low-density lipoprotein (MM-LDL) is a potent atherogenic lipoprotein. We analyzed the effects of MM-LDL on brain capillary endothelial expression of plasminogen activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (tPA), and thrombomodulin (TM). Cultured bovine brain capillary endothelial cells (BEC) incubated with MM-LDL (25 μg/ml) for 24 h showed increased PAI-1 mRNA levels by approximately seven-fold, while tPA and TM mRNA levels were reduced by 84% and 75%, respectively. Moreover, PAI-1 protein levels increased two-fold (16.8±7.6 vs. 7.6±2.1 ng/ml, p<0.05), whereas tPA protein levels decreased by 45% (1.3±0.5 ng/ml vs. 2.3±0.7 ng/ml, p<0.05), and TM protein level decreased by 40%. Following incubation with MM-LDL, PAI-1 activity was increased 35% (18.4±5.0 vs. 24.8±5.2 AU/ml, p<0.05), while TM activity was decreased by 30%. MM-LDL therefore has substantial pro-thrombotic effects on brain capillary endothelial cells, reducing both endothelial fibrinolytic capacity (downregulating tPA while upregulating PAI-1) and anticoagulant function (downregulating TM). These results suggest that MM-LDL may contribute to thrombus formation in the brain.

Mechanisms of Hypoxic Regulation of Plasminogen Activator Inhibitor-1 Gene Expression in Keloid Fibroblasts

Keloids are an excessive accumulation of extracellular matrix. Although numerous studies have shown elevated plasminogen activator inhibitor-1 (PAI-1) levels in keloid fibroblasts compared with those of normal skin. Their specific mechanisms involved in the differential expression of PAI-1 in these cell types. In this study, the upregulation of PAI-1 expression is demonstrated in keloid tissues and their derived dermal fibroblasts, attesting to the persistence, if any, of fundamental differences between in vivo and in vitro paradigms. We further examined the mechanisms involved in hypoxia-induced regulation of PAI-1 gene in dermal fibroblast derived from keloid lesions and associated clinically normal peripheral skins from the same patient. Primary cultures were exposed to an environmental hypoxia or desferroxamine. We found that the hypoxia-induced elevation of PAI-1 gene appears to be regulated at both transcriptional and post-transcriptional levels in keloid fibroblasts. Furthermore, our results showed a consistent elevation of HIF-1alpha protein level in keloid tissues compared with their normal peripheral skin controls, implying a potential role as a biomarker for local skin hypoxia. Treatment with antisense oligonucleotides against hypoxia-inducible factor 1alpha (HIF-1alpha) led to the downregulation of steady-state levels of PAI-1 mRNA under both normoxic and hypoxic conditions. Conceivably, our results suggest that HIF-1alpha may be a novel therapeutic target to modulate the scar fibrosis process.

Rosiglitazone inhibits the insulin-mediated increase in PAI-1 secretion in human abdominal subcutaneous adipocytes.

The aim of this study was to investigate the effect of insulin and an insulin-sensitizing agent, rosiglitazone (RSG), on the production of plasminogen-activator inhibitor-1 (PAI-1) in isolated subcutaneous abdominal adipocytes. Human tissue-type plasminogen activator (t-PA) was also measured to assess changes in overall thrombotic risk. The mean depot-specific expression of PAI-1 and t-PA mRNA (n = 42) in subcutaneous abdominal (n = 21), omental (n = 10) and thigh (n = 11) adipose tissue depots was examined. Furthermore, subcutaneous adipocytes were treated with insulin, RSG and insulin in combination with RSG (10-8 m) for 48 h. Conditioned media were collected and enzyme-linked immunosorbent assays performed for PAI-1 and t-PA (n = 12) antigen. PAI-1 and t-PA mRNA levels were also assessed. PAI-1 mRNA levels were significantly higher in subcutaneous and omental abdominal tissue than in thigh fat (p = 0.037 and p = 0.014). No change in t-PA mRNA expression between the adipose tissue depots was observed. Insulin stimulated PAI-1 protein secretion in a concentration-dependent manner in adipocytes (control: 68.3 +/- 1.2 ng/ml (s.e.m.); 10 nm insulin: 73.7 +/- 3.8 ng/ml upward arrow; 100 nm insulin: 86.8 +/- 4.1 ng/ml upward arrow **; 1000 nm insulin: 102.0 +/- 4.8 ng/ml upward arrow ***; **p < 0.01, ***p < 0.001). In contrast, insulin + RSG (10-8 m) reduced PAI-1 production relative to insulin alone (***p < 0.001), whilst RSG alone reduced PAI-1 protein secretion in a concentration-dependent manner (RSG at 10-10 m: 50.4 +/- 2.87 ng/ml downward arrow ***; RSG at 10-5 m: 30.3 +/- 2.0 ng/ml downward arrow ***; p < 0.001). No difference was observed between control and treatments for t-PA secretion (range 7-11 ng/ml). Insulin stimulated PAI-1 secretion, whilst RSG reduced both PAI-1 secretion alone and in combination with insulin. These data suggest that adipose tissue may contribute significantly to the elevated circulating PAI-1 in obesity. Therefore, RSG's effects on PAI-1 production in adipose tissue may contribute to the fall in circulating PAI-1 levels observed in patients receiving RSG therapy.

Astrocyte regulation of human brain capillary endothelial fibrinolysis

Introduction: Astrocytes are known to regulate a wide variety of brain endothelial cell functions. Prior work, using a mixed species co-culture system, has shown astrocyte regulation of brain capillary endothelial expression of key hemostasis factors tissue plasminogen activator (tPA) and its inhibitor, plasminogen activator inhibitor-1 (PAI-1). The purpose of this study is to define the fibrinolytic regulatory role of human astrocytes on human brain capillary endothelial cells. Materials and methods: We used a blood–brain barrier model consisting of human astrocytes grown on transwell membrane inserts and co-cultured with human brain capillary endothelial cells. Following 48 h co-culture, we analyzed both endothelial mono-cultures and astrocyte-endothelial co-cultures for expression of tPA and PAI-1 mRNA, protein, and activity. Results and conclusions: There were significant changes for both tPA and PAI-1 mRNA:tPA mRNA levels were decreased in co-cultures (55±16% of mono-cultures, p<0.0005) and PAI-1 mRNA levels were increased 144±38%, compared to mono-cultures ( p<0.005). Co-cultures produced a 54% reduction in tPA protein (12.7±3.8 vs. 27.5±7.1 ng/ml, p<0.005) and a 24% increase in PAI-1 protein (117.5±3.2 vs. 94.9±5.9 ng/ml, p<0.0005). TGF-β neutralizing antibody attenuated the observed changes in both tPA and PAI-1. These data indicate that human astrocytes regulate human brain capillary fibrinolysis in vitro by inhibiting tPA and enhancing PAI-1 expression. This regulation is mediated, in part, by transforming growth factor-β. Our findings provide further evidence for the role of astrocytes in brain-specific hemostasis regulation.

Reactive oxygen species modulate HIF-1 mediated PAI-1 expression: involvement of the GTPase Rac1

SummaryThe hypoxia-inducible transcription factor HIF-1 mediates upregulation of plasminogen activator inhibitor-1 (PAI-1) expression under hypoxia. Reactive oxygen species (ROS) have also been implicated in PAI-1 gene expression. However, the role of ROS in HIF-1-mediated regulation of PAI-1 is not clear. We therefore investigated the role of the GTPase Rac1 which modulates ROS production in the pathway leading to HIF-1 and PAI-1 induction.Overexpression of constitutively activated (RacG12V) or dominant-negative (RacT17N) Rac1 increased or decreased, respectively, ROS production. In RacG12V-expressing cells, PAI-1 mRNA levels as well as HIF-1α nuclear presence were reduced under normoxia and hypoxia whereas expression of RacT17N resulted in opposite effects. Treatment with the antioxidant pyrrolidinedithiocarbamate or coexpression of the redox factor-1 restored HIF-1 and PAI-1 promoter activity in RacG12V-cells. In contrast, NFκB activation was enhanced in RacG12V-cells, but abolished by RacT17N. Thus, these findings suggest a mechanism explaining modified fibrinolysis and tissue remodeling in an oxidized environment.

Regulation of the hypoxia-dependent plasminogen activator inhibitor 1 expression by MAP kinases

Mitogen-activated protein kinases (MAPKs) and protein kinase B (PKB) mediate growth and stress signals and have been implicated in the hypoxic response. Under hypoxic conditions, the expression of plasminogen activator inhibitor-1 (PAI-1) is mainly controlled by the hypoxia-inducible factor HIF-1. However, the role of MAPKs and PKB in HIF-1-mediated PAI-1 regulation is not clear. Treatment with the p38 inhibitor SB203580 and the PI3K inhibitor LY294002, but not with the MEK1 inhibitor PD98059, abrogated hypoxia-dependent PAI-1 induction in HepG2 cells. Consistently, overexpression of PKB or of the p38 upstream kinases MKK6 and MKK3 and of JNK, but not of ERK, enhanced PAI-1 mRNA levels. In MKK3-, MKK6- and PKB-expressing cells luciferase (Luc) activities from a hypoxia-inducible PAI-1-Luc construct or from a HIF-dependent Luc construct and, concomitantly, HIF-1alpha protein levels were enhanced. These findings indicate that p38- and PKB-dependent signalling pathways contribute to enhanced PAI-1 levels in the hypoxic response.

Biphasic regulation of plasminogen activator/inhibitor by LDL in mesangial cells.

Lipid abnormalities and dysregulation of the plasminogen activator (PA)/plasmin system may be involved in the development of glomerulosclerosis. We investigated the effects of low-density lipoprotein (LDL) on PA inhibitor-1 (PAI-1), urokinase-type PA (uPA), and tissue-type PA (tPA) in relationship to protein kinase C (PKC) in cultured human mesangial cells (HMC). LDL (200 microg/ml) induced two peaks of PKC activation at hours 0.25 and 6, with translocation of PKC-alpha, -beta(1), and -delta from cytosol to the membrane. The second increase in PKC activity gradually decreased to the control value by hour 18. LDL downregulated 2.4-kb PAI-1, uPA, and tPA mRNA expression within 6 h of incubation with HMC. On the other hand, after 12-48 h, LDL-treated cells showed a significant increase in PAI-1, tPA, and uPA mRNA levels. LDL induced up to a twofold increase in PAI-1 antigen levels in the extracellular matrix of HMC after 24-48 h as well as increased PA inhibitory activity in the culture medium. Analysis of the adhesion plaques from cells incubated with LDL for 48 h by zymography showed increased intensity of lysis near molecular weights of approximately 55,000 and 100,000. LDL slightly increased tPA release at hours 24 and 48 but did not increase PA activity in culture medium. The stimulatory effects of LDL on PAI-1, tPA, and uPA gene regulation in HMC were blocked by the inhibition of PKC using GF-109203X 12 h after treatment with LDL or downregulation of PKC using phorbol myristate acetate. In summary, LDL regulates PAI-1, uPA, and tPA in biphasic patterns in HMC, and the upregulation of PAI-1, uPA, and tPA after long-term LDL exposure seems to be mediated by a delayed PKC activation associated with an increased PA inhibitory activity. These results suggest that LDL, after prolonged incubations with HMC, causes a PA/inhibitor imbalance favoring accumulation of matrix.

Gene expression profile analysis of rheumatoid synovial fibroblast cultures revealing the overexpression of genes responsible for tumor-like growth of rheumatoid synovium

To elucidate the aberrant growth properties of rheumatoid synoviocytes, we have examined the gene expression profile of rheumatoid synovial fibroblasts (RSFs) and compared with that of normal synovial fibroblasts (NSF). Gene expression profile analysis was conducted with synoviocyte cultures obtained from five rheumatoid arthritis (RA) patients and five control cases using a commercial cDNA array containing the defined 588 cancer-related genes. The results were confirmed by real-time RT-PCR. Gene expression levels for the platelet-derived growth factor receptor α (PDGFRα), plasminogen activator inhibitor-1 (PAI-1), and stromal cell derived factor 1A (SDF1A) are constitutively augmented in RSF compared with NSF. The mRNA levels of PDGFRα, PAI-1, and SDF1A in RSF over NSF were 4.6-, 14-, and 2.8-fold, respectively, by real-time RT-PCR. In fact, we found that RSFs showed greater sensitivity to the cell proliferative effect of PDGF. T his aberrant gene expression profile suggests that RSF may have retained the premature phenotype of primordial synoviocytes.