Plasminogen Activator Inhibitor-1 Transcription Research Articles

ERK contributes to the effects of Smad signaling on oxidized LDL‐induced PAI‐1 experssion in human mesangial cells

Hypercholesterolemia and lipoprotein abnormalities are risk factors for the progression of glomerular injury to glomerulosclerosis. Oxidized Low-Density Lipoprotein (Ox-LDL) stimulates plasminogen activator inhibitor-1 (PAI-1) expression in human mesangial cells mediated by transforming growth factor-beta (TGF-beta)/Smad signaling pathway. TGF-beta activates extracellular signal-regulated kinase (ERK) in mesangial cells, and ERK is involved in activation of Smad2/3. This study examines whether an interaction exists between Ox-LDL-induced TGF-beta/Smad signaling pathways and ERK activation leading to PAI-1 transcription in human mesangial cells. Ox-LDL (50 microg/mL) induced an acute increase in ERK activity within 15 min, which decreased to control value at 2 h. Incubation with anti-TGF-beta or SB-431542, an inhibitor of the TGF-beta type I receptor, along with Ox-LDL, inhibited the expected increase in ERK phosphorylation. Treatment with PD98059 or UO126, mitogen-activated ERK-activating kinase 1/2 inhibitors, significantly inhibited the Ox-LDL-induced increase in PAI-1 mRNA and nuclear Smad3 expression, DNA/protein complex formation, and PAI-1 promoter activity. These results suggest that phosphorylation of ERK is induced by Ox-LDL through the induction of the TGF-beta signaling pathway and that activated ERK, in turn, participates in the Ox-LDL-induced Smad3 activation and subsequent PAI-1 gene expression in mesangial cells. Thus, in patients with chronic renal disease and persistent hypercholesterolemia, ERK activation by Ox-LDL might contribute to mesangial ECM accumulation and subsequently renal fibrosis due to interaction between Ox-LDL and the Smad signaling pathway.

Molecular regulation of the PAI‐1 gene by hypoxia: contributions of Egr‐1, HIF‐1 α, and C/EBPα

Hypoxia, as occurs during tissue ischemia, tips the natural anticoagulant/procoagulant balance of the endovascular wall to favor activation of coagulation. Plasminogen activator inhibitor-1 (PAI-1) is an important factor suppressing fibrinolysis under conditions of low oxygen tension. We previously reported that hypoxia induced PAI-1 mRNA and antigen expression in murine macrophages secondary to increased de novo transcription as well as increased mRNA stability. We now show in RAW264.7 murine macrophages that the transcription factors early growth response gene-1 (Egr-1), hypoxia-inducible factor-1alpha (HIF-1alpha), and CCAAT/enhancer binding protein alpha (C/EBPalpha) are quickly activated in hypoxia and are responsible for transcription and expression of PAI-1. Murine PAI-1 promoter constructs, including Egr, HIF-1alpha, and/or C/EBPalpha binding sites, were transfected into RAW 264.7 murine macrophages. To identify the relative importance of each of these putative hypoxia-responsive elements, cells were exposed to normobaric hypoxia, and transcriptional activity was recorded. At 16 h of hypoxic exposure, murine PAI-1 promoter deletion constructs that included Egr, HIF-1alpha, and/or C/EBPalpha binding sites demonstrated increased transcriptional activity. Mutation of each of these three murine PAI-1 promoter sites (or a combination of them) resulted in a marked reduction in hypoxia sensitivity as detected by transcriptional analysis. Functional data obtained using 32P-labeled Egr, HIF-1alpha response element (HRE), and C/EBPalpha oligonucleotides revealed induction of DNA binding activity in nuclear extracts from hypoxic RAW cells, with supershift analysis confirming activation of Egr-1, HIF-1alpha, or C/EBPalpha. ChIP analysis confirmed the authenticity of these interactions as each of these transcription factors binds to chromatin under hypoxic conditions. Further, the induction of PAI-1 by Egr-1, HIF-1alpha, or C/EBPalpha was replicated in primary peritoneal macrophages. These data suggest that although HIF-1alpha appears to dominate the PAI-1 transcriptional response in hypoxia, Egr-1 and C/EBPalpha greatly augment this response and can do so independent of HIF-1alpha or each other. These studies are relevant to ischemic up-regulation of the PAI-1 gene and consequent accrual of microvascular thrombus under ischemic conditions.

Ki26894, a novel transforming growth factor‐β type I receptor kinase inhibitor, inhibits in vitro invasion and in vivo bone metastasis of a human breast cancer cell line

Transforming growth factor (TGF)-beta signaling has been shown to promote tumor growth and metastasis in advanced cancer. Use of inhibitors of TGF-beta signaling may thus be a novel strategy for treatment of patients with such cancers. In this study, we investigated the effects of a novel TGF-beta type I receptor (TbetaR-I) kinase inhibitor, Ki26894, on bone metastasis of a highly bone-metastatic variant of human breast cancer MDA-MB-231 cells, termed MDA-MB-231-5a-D (MDA-231-D). Ki26894 blocked TGF-beta signaling in MDA-231-D cells, as detected by suppression of phosphorylation of Smad2 and inhibition of TGF-beta-responsive reporter activity. Moreover, Ki26894 decreased the motility and the invasion of MDA-231-D cells induced by TGF-beta in vitro. Ki26894 also suppressed transcription of plasminogen activator inhibitor-1 (PAI-1), parathyroid hormone-related protein (PTHrP), and interleukin-11 (IL-11) mRNA of MDA-231-D cells, which were stimulated by TGF-beta. X-ray radiography revealed that systemic Ki26894 treatment initiated 1 day before the inoculation of MDA-231-D cells into the left ventricle of BALB/cnu/nu female mice resulted in decreased bone metastasis of breast cancer cells. Moreover, Ki26894 prolonged the survival of mice inoculated with MDA-231-D cells compared to vehicle-treated mice. These findings suggest that TbetaR-I kinase inhibitors such as Ki26894 may be useful for blocking the progression of advanced cancers.

Functional Plasminogen Activator Inhibitor-1 Gene Variants and Breast Cancer Survival

Plasminogen activator inhibitor-1 (PAI-1) plays an important role in cancer invasion and metastasis. A common polymorphism (4G/5G) in the promoter region of the PAI-1 gene has been reported to influence transcription and plasma levels of PAI-1. We evaluated the association between PAI-1 4G/5G polymorphism and breast cancer survival in a population-based cohort of breast cancer patients. Included in this analysis were 1,083 Chinese women diagnosed with stage 0 to III primary breast cancer at age 25 to 64 years who were recruited between 1996 and 1998 for the Shanghai Breast Cancer Study and followed for a median of 5.2 years. The Kaplan-Meier method and Cox model were used to evaluate the genotype and survival association. After adjustment for known prognostic factors for breast cancer, patients homozygous for the 4G allele had significantly poorer disease-free survival [hazard ratio (HR), 1.7; 95% confidence interval (95% CI), 1.1-2.4] and overall survival (HR, 1.5; 95% CI, 1.0-2.3) than those homozygous for the 5G allele. The association was more evident in patients with advanced disease. The HRs (95% CI) were 3.5 (1.4-9.0) for disease-free survival and 3.1 (1.1-8.3) for overall survival in stage III patients. The PAI-1 4G/5G polymorphism may be a prognostic marker for young and middle-aged Chinese breast cancer patients.

ERK contributes to the effects of Smad signaling on oxidized LDL-induced PAI-1 expression in human mesangial cells

Oxidized low-density lipoprotein (Ox-LDL) stimulates plasminogen activator inhibitor-1 (PAI-1) expression in human mesangial cells mediated by transforming growth factor-beta (TGF-beta)/Smad signaling pathway. TGF-beta activates extracellular signal-regulated kinase (ERK) in mesangial cells, and ERK is involved in activation of Smad2/3. This study examines whether an interaction exists between Ox-LDL-induced TGF-beta/Smad signaling pathways and ERK activation leading to PAI-1 transcription in human mesangial cells. Ox-LDL (50 microg/mL) induced an acute increase in ERK activity within 15 min, which decreased to control value at 2 h. Incubation with anti-TGF-beta or SB-431542, an inhibitor of the TGF-beta type I receptor, along with Ox-LDL, inhibited the expected increase in ERK phosphorylation. Treatment with PD98059 or UO126, mitogen-activated ERK-activating kinase 1/2 inhibitors, significantly inhibited the Ox-LDL-induced increase in PAI-1 mRNA and nuclear Smad3 expression, DNA/protein complex formation, and PAI-1 promoter activity. These results suggest that phosphorylation of ERK is induced by Ox-LDL through the induction of the TGF-beta signaling pathway and that activated ERK, in turn, participates in the Ox-LDL-induced Smad3 activation and subsequent PAI-1 gene expression in mesangial cells.

Shear stress induces hepatocytePAI-1gene expression through cooperative Sp1/Ets-1 activation of transcription

Partial hepatectomy causes hemodynamic changes that increase portal blood flow in the remaining lobe, where the expression of immediate-early genes, including plasminogen activator inhibitor-1 (PAI-1), is induced. We hypothesized that a hyperdynamic circulatory state occurring in the remaining lobe induces immediate-early gene expression. In this study, we investigated whether the mechanical force generated by flowing blood, shear stress, induces PAI-1 expression in hepatocytes. When cultured rat hepatocytes were exposed to flow, PAI-1 mRNA levels began to increase within 3 h, peaked at levels significantly higher than the static control levels, and then gradually decreased. The flow-induced PAI-1 expression was shear stress dependent rather than shear rate dependent and accompanied by increased hepatocyte production of PAI-1 protein. Shear stress increased PAI-1 transcription but did not affect PAI-1 mRNA stability. Functional analysis of the 2.1-kb PAI-1 5'-promoter indicated that a 278-bp segment containing transcription factor Sp1 and Ets-1 consensus sequences was critical to the shear stress-dependent increase of PAI-1 transcription. Mutations of both the Sp1 and Ets-1 consensus sequences, but not of either one alone, markedly prevented basal PAI-1 transcription and abolished the response of the PAI-1 promoter to shear stress. EMSA and chromatin immunoprecipitation assays showed binding of Sp1 and Ets-1 to each consensus sequence under static conditions, which increased in response to shear stress. In conclusion, hepatocyte PAI-1 expression is flow sensitive and transcriptionally regulated by shear stress via cooperative interactions between Sp1 and Ets-1.

Effects of oxidized and glycated low-density lipoproteins on transcription and secretion of plasminogen activator inhibitor-1 in vascular endothelial cells

Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of fibrinolysis. Elevated levels of PAI-1 were frequently detected in patients with coronary artery disease (CAD) or diabetes. Low-density lipoprotein (LDL) is a classical risk factor of CAD. Oxidation and glycation increase the atherogenecity of LDL. Previous studies demonstrated that oxidized LDL (oxLDL) or glycated LDL (gly-LDL) increased the release of PAI-1 from endothelial cells (ECs). The present study examined the effects of oxLDL and gly-LDL on the transcription, expression, secretion, and subcellular distribution of PAI-1 in cultured human ECs. Treatment with LDL significantly increased the promoter activity, mRNA level, and the release of PAI-1 from ECs by two- to threefold compared to controls. Oxidation or glycation significantly enhanced the effects of LDL on PAI-1 production in ECs compared to LDL (four- to fivefold vs. controls). No significant differences were detected between the effects of oxLDL and gly-LDL. Abundant PAI-1 antigens were detected in the perinuclear region of ECs and overlapped with giantin, a marker of Golgi apparatus. Treatment with brefeldin A disturbed the stack structure of Golgi apparatus and almost completely inhibited the release of PAI-1 from ECs induced by the lipoproteins and at basal conditions. The results suggest that oxidation and glycation enhanced the effects of LDL on the production of PAI-1 in ECs through increasing the transcription of PAI-1. Intact Golgi apparatus is required for PAI-1 generation from ECs induced by LDL or its modified forms.

Radiation and Transforming Growth Factor-β Cooperate in Transcriptional Activation of the Profibrotic Plasminogen Activator Inhibitor-1 Gene

Radiation-induced fibrosis is an important side effect in the treatment of cancer. Profibrotic proteins, such as plasminogen activator inhibitor-1 (PAI-1), transforming growth factor-beta (TGF-beta), and tissue type inhibitor of metalloproteinases-1 (Timp-1), are thought to play major roles in the development of fibrosis via the modulation of extracellular matrix integrity. We did a detailed analysis of transcriptional activation of these profibrotic genes by radiation and TGF-beta. Irradiation of HepG2 cells led to a high increase in PAI-1 mRNA levels and a mild increase in Timp-1 mRNA levels. In contrast, TGF-beta1 and Smad7 were not increased. Radiation and TGF-beta showed strong cooperative effects in transcription of the PAI-1 gene. The TGF-beta1 gene showed a mild cooperative activation, whereas Timp-1 and Smad7 were not cooperatively activated by radiation and TGF-beta. Analysis using the proximal 800 bp of the human PAI-1 promoter revealed a dose-dependent increase of PAI-1 levels between 2 and 32 Gy gamma-rays that was independent of latent TGF-beta activation. Subsequent site-directed mutagenesis of the PAI-1 promoter revealed that mutation of a p53-binding element abolished radiation-induced PAI-1 transcription. In line with this, PAI-1 was not activated in p53-null Hep3B cells, indicating that p53 underlies the radiation-induced PAI-1 activation and the cooperativity with the TGF-beta/Smad pathway. Together, these data show that radiation and TGF-beta activate PAI-1 via partially nonoverlapping signaling cascades that in concert synergize on PAI-1 transcription. This may play a role in patient-to-patient variations in susceptibility toward fibrosis after radiotherapy.

Synergistic effect of hypoxia and TNF-α on production of PAI-1 in human proximal renal tubular cells

Chronic hypoxia has been newly proposed as a common mechanism of tubulointerstitial fibrosis in the progression of various chronic inflammatory renal diseases, where plasminogen activator inhibitor-1 (PAI-1) plays an important role in the accumulation of extracellular matrix (ECM) through inhibition of plasmin-dependent ECM degradation. In the present study, we investigated the presence of PAI-1 in renal tubular cells by immunostaining renal biopsy samples. We also closely examined the effects of hypoxia and tumor necrosis factor-alpha (TNF-alpha) on PAI-1 expression in cultured human proximal renal tubular cells (HPTECs). Confluent cells growth-arrested in Dulbecco's modified Eagle's medium (DMEM) for 24 hours were exposed to hypoxia (1% O(2)) and/or TNF-alpha at 10 ng/mL for up to 48 hours. Amounts of PAI-1 protein and mRNA after stimulation were measured by enzyme-linked immunosorbent assay (ELISA) and TaqMan quantitative polymerase chain reaction (PCR) or cDNA array analysis, respectively, and compared to those in cells incubated under control conditions (18% O(2) without TNF-alpha). Hypoxia-inducible factor-1alpha (HIF-1alpha) was demonstrated by immunoblot and immunofluorescence analyses. Human PAI-1 promoter activity was estimated by luciferase reporter gene assay. In crescentic glomerulonephritis, clusters of proximal tubules were specifically stained for PAI-1. cDNA array analysis identified PAI-1 as a major gene highly induced by hypoxia in HPTECs. Treatment of 24 hours with hypoxia, TNF-alpha, and their combination induced a 2.8-fold, a 1.8-fold, and a 4.6-fold increase in PAI-1 protein secretion, and produced a 3.6-fold, a 3.3-fold, and a 12.1-fold increase at the PAI-1 mRNA level, respectively. Immunoblot analysis and immunocytochemistry revealed that hypoxia-inducible factor-1alpha (HIF-1alpha) was markedly accumulated in the cell lysates and exclusively translocated to nuclei after 16 hours' exposure of HPTECs to hypoxia but not to TNF-alpha. Luciferase reporter gene assay showed that hypoxia, TNF-alpha, and their combination increased PAI-1 transcription activity by 1.8-fold, 1.4-fold, and 2.2-fold, respectively. A dominant-negative form of HIF-1alpha significantly suppressed PAI-1 transcription activity induced by hypoxia. Inhibition of nuclear factor-kappaB (NF-kappaB) caused a moderate decrease in PAI-1 production under hypoxia. Hypoxia induces PAI-1 expression via remarkable nuclear accumulation of HIF-1alpha and partially via NF-kappaB activation in HPTECs. TNF-alpha can synergistically enhance this hypoxia-induced PAI-1 expression.

Transcriptional regulation of plasminogen activator inhibitor-1 by transforming growth factor-β, activin A and microphthalmia-associated transcription factor

Plasminogen activator inhibitor-1 ( PAI-1) is a key molecule that regulates turnover of the extracellular matrix. In the present study, we characterized PAI-1 gene expression in mast cells and melanocytes. In bone marrow-derived cultured mast cells, up-regulation of the PAI-1 gene was observed upon treatment with TGF-β 1, and was regulated at the transcriptional level. Microphthalmia-associated transcription factor (MITF), a member of the basic helix–loop–helix leucine zipper family of tissue-specific transcription factors predominantly expressed in mast cells, melanocytes and osteoclasts, also stimulated PAI-1 gene transcription, and TGF-β 1 did not increase PAI-1 mRNA levels in mast cells from mi/ mi mice expressing dominant-negative MITF. MITF isoforms regulated TGF-β 1-induced transcription of PAI-1 differently; MITF-E-mediated transcription was further increased by TGF-β 1, whereas transcriptional activation by TGF-β 1 was blocked by MITF-M or MITF-mc expression. In contrast, activin A, another member of the TGF-β family, enhanced transcription induced by MITF-M, as well as by MITF-E, although MITF-mc blocked activin A-induced transcription of PAI-1. Different regulation of PAI-1 gene expression upon TGF-β 1 and activin A treatment was also detected in B16 melanocytes; TGF-β 1 transiently increased the PAI-1 mRNA level, whereas activin A had prolonged effects on up-regulation of PAI-1. Our results on the control of PAI-1 gene expression by MITF isoforms, TGF-β 1 and activin A suggest that discrete regulation of the plasminogen activator system occurs in a cell type-specific manner.

Oxidized LDL activates PAI-1 transcription through autocrine activation of TGF-β signaling in mesangial cells

Lipid abnormalities and oxidative stress may be involved in the development of glomerulosclerosis. Plasminogen activator inhibitor-1 (PAI-1) is a component of extracellular matrix (ECM) and target gene of transforming growth factor-beta (TGF-beta). Smad proteins play a key role in TGF-beta signaling, and Smad binding CAGA boxes are present in the PAI-1 promoter. This study examined whether oxidized low-density lipoprotein (Ox-LDL) activates PAI-1 transcription in human mesangial cells, mediated by increased Smad/DNA interactions. Quiescent HMC were incubated with 50 microg/mL of Cu(++)-catalyzed Ox-LDL for 15 minutes to 4 hours, and the effects of Ox-LDL on TGF-beta1 and PAI-1 mRNA expression, PAI-1 promoter activity, and DNA binding activity of Smad proteins were examined. Ox-LDL induced TGF-beta1 and PAI-1 mRNA expression. Ox-LDL increased the transiently transfected PAI-1 promoter activity as compared with controls to 3.9-fold. Ox-LDL-treated cells increased Smad3 protein levels two times the control levels in the nuclei. Electrophoretic mobility shift assay (EMSA) performed using a CAGA sequence probe and nuclear extracts showed that Ox-LDL increased DNA/protein complexes. When nuclear extracts were preincubated with 100 molar excess of unlabeled CAGA oligonucleotide or SB-431542, an inhibitor of the TGF-beta type I receptor, the formation of complex was prevented. The DNA binding protein was shown to be Smad3 by antibody supershift. Transfection of phosphorothioate CAGA oligonucleotides, which compete with the CAGA-containing PAI-1 promoter for Smad3 binding, inhibited the Ox-LDL-induced PAI-1 mRNA expression. Cotransfection of phosphorothioate CAGA oligonucleotides with PAI-1 reporter vector also blocked the Ox-LDL-induced PAI-1 promoter activity. These results suggest that Ox-LDL activates TGF-beta/Smad signaling to stimulate PAI-1 transcription in human mesangial cells. Thus, progression of glomerular disease may be promoted by PAI-1 up-regulation in human mesangial cells mediated by the Ox-LDL-induced TGF-beta/Smad signaling pathways.

Plasminogen activator inhibitor-1 (PAI-1) expression in vascular smooth muscle cells involves a SRC/MEK/USF signal cascade

Background High PAI-1 levels associate with increased cardiovascular disease risk. PAI-1 targeting may be an important approach to the therapy of vascular disorders. Methods Pathway-specific pharmacologic/molecular agents were used to define signaling events associated with PAI-1 expression. Results MEK (PD98059, U0126) or src family (PP1) kinase inhibitors attenuated growth factor-stimulated PAI-1 transcription in smooth muscle cells. PP1 blocked ERK1/2 activation suggesting that the required src kinase is upstream of ERK1/2. Transfection of a dominant-negative pp60c-src construct, moreover, reduced PAI-1 levels to that of PP1-treated controls implicating pp60c-src as the involved src kinase. An E box motif in the PAI-1 promoter was determined to be a USF-1 binding site. Mutation of this site or transfection of dominant-negative USF-1 constructs attenuated PAI-1 expression in smooth muscle cells. Nuclear USF-1 was determined to be a phosphorylation target of translocated ERK1/2 by both in situ co-localization microscopy and co-immunoprecipitation. Conclusions A major phenotypic characteristic of “activated” smooth muscle cells (i.e., transcription of fibrosis-associated target genes[e.g., PAI-1]) requires pp60c-src kinase activity and MEK signaling and involves activation of the USF transcription factor likely by ERK family MAP kinases. Pharmacologic or genetically-targeted manipulation of this pathway may have therapeutic usefulness in treatment of vascular disease. Clinical Pharmacology & Therapeutics (2005) 77, P11–P11; doi: 10.1016/j.clpt.2004.11.043

Vascular Endothelial Cell Growth Factor Attenuates Actions of Transforming Growth Factor-β in Human Endothelial Cells

Because vascular endothelial cell growth factor (VEGF) and transforming growth factor-beta (TGF-beta) are both involved in cellular growth and differentiation, we examined whether VEGF modifies TGF-beta signaling cascade in human umbilical cord vein endothelial cells (HUVEC). Production of plasminogen activator inhibitor-1 (PAI-1), which is under the specific control of TGF-beta, was strongly enhanced (3.5-fold) by TGF-beta treatment. Remarkably, physiological concentration of VEGF (30 nm) profoundly (by 60%) attenuated the TGF-beta stimulation of PAI-1 production without an effect on the basal PAI-1 production. In HUVECs transiently transfected with an expression construct containing a PAI-1 promoter fused to luciferase reporter gene, TGF-beta-stimulation of transcription of PAI-1 was clearly (by 60%) inhibited by VEGF. TGF-beta phosphorylation of Smad2/3, an obligatory step of intracellular TGF-beta signaling, was also suppressed by VEGF. VEGF attenuation of TGF-beta action was also demonstrated in two other endothelial cell lines. In conclusion, VEGF attenuates TGF-beta action in the human endothelial cell, specifically at the level of transcription of PAI-1 gene and Smad2/3 phosphorylation.

Glycated albumin activates PAI-1 transcription through Smad DNA binding sites in mesangial cells.

Amadori-modified glycated albumin stimulates extracellular matrix and transforming growth factor-beta (TGF-beta) expression in cultured mesangial cells. Smad proteins transduce the TGF-beta-mediated signal, and Smad-binding CAGA sequences are present in the plasminogen activator inhibitor-1 (PAI-1) promoter. This study examined whether glycated albumin induces PAI-1 transcription in human mesangial cells (HMC) through Smad-binding sites in the PAI-1 promoter. Quiescent HMC were exposed to 200 microg/ml bovine serum albumin (BSA) or glycated BSA (Gly-BSA) for 12-72 h. At 24 h, Gly-BSA stimulated TGF-beta1 and PAI-1 mRNA expression in HMC to 1.8 and 3.2 times that in the BSA-treated control cells. Gly-BSA also activated the PAI-1 promoter luciferase activity 2.3-fold. Gly-BSA-treated cells enhanced Smad2 and Smad3 protein levels 2.5 times the control levels in the nuclei. An electrophoretic mobility shift assay performed using CAGA sequences as a probe showed that Gly-BSA increased DNA/protein complexes. When nuclear extracts were preincubated with 100-fold molar excess of unlabeled CAGA oligonucleotide, the formation of complex was prevented. The DNA-binding protein was shown to be Smad3 by antibody supershift. Transfection of phosphorothioate CAGA oligonucleotide, a CAGA antisense analog, inhibited Gly-BSA-induced PAI-1 mRNA expression. Cotransfection of phosphorothioate CAGA oligonucleotides with PAI-1 reporter vector also blocked Gly-BSA-induced PAI-1 promoter luciferase activity. These results indicate that Gly-BSA increases DNA binding activity of Smad3 and that it stimulates PAI-1 transcription through Smad-binding CAGA sequences in the PAI-1 promoter in HMC. Thus progression of diabetic nephropathy may be promoted by PAI-1 upregulation mediated by the glycated albumin-induced Smad/DNA interactions.

De novo synthesis of diacylglycerol in endothelium may mediate the association between PAI-1 and the insulin resistance syndrome

Increased free fatty acid flux, giving rise to increased de novo synthesis of diacylglycerol (DAG) and activation of protein kinase C (PKC) in vascular endothelium, may be largely responsible for the endotheliopathy and increased vascular risk associated with insulin resistance syndrome. This mechanism may also mediate, in large part, the increase in plasminogen activator inhibitor-1 (PAI-1) observed in this syndrome. PKC activation promotes transcription of PAI-1 in endothelial cells and other tissues, apparently by boosting the activity of Sp1 transcription factors that bind to the PAI-1 promoter. Plasma PAI-1 correlates inversely with the ability of insulin infusion to suppress free fatty acid levels. Moreover, infusion of triglycerides with heparin – inducing a marked increase in free fatty acids – has been shown to induce a rapid increase in plasma PAI-1. Alternatively, hyperinsulinemia and hypertriglyceridemia have been suggested as mediators of PAI-1 excess in insulin resistance, inasmuch as insulin and VLDL can stimulate PAI-1 production in cell cultures. However, plasma PAI-1 tends to decline in response to hyperinsulinemic clamps and insulin treatment of type 2 diabetes, and gemfibrozil treatment of hypertriglyceridemia does not decrease PAI-1 – suggesting that elevations of insulin or triglycerides are not likely to mediate PAI-1 excess in vivo. Hypertrophied adipose mass can secrete PAI-1, and is likely to contribute to the plasma PAI-1 pool in obese insulin-resistant subjects, but current evidence suggests that this is not likely to be the primary source of the elevated plasma PAI-1 in insulin resistance syndrome. Plasma PAI-1 can be decreased in insulin resistant subjects by improving adipocyte insulin sensitivity (with weight loss and thiazolidinediones), by consuming a very-low-fat diet that minimizes postprandial free fatty acid flux, and by administering activators of AMP-activated kinase (e.g., metformin), which can be expected to lessen tissue DAG synthesis.

Interrelationships between the fibrinolytic system and lipoproteins in the pathogenesis of coronary atherosclerosis

The fibrinolytic system is comprised of a series of serine proteases and serine protease inhibitors which are involved in the dissolution of fibrin in the vascular lumen, but also in the migration of cells and in the remodeling of the extracellular matrix of the vascular wall. The transcription, expression and degradation of the various fibrinolytic enzymes by cells in the vascular wall is influenced by lipoproteins and this interrelationship may play a significant role in the development of the atherosclerotic plaque: the transcription of plasminogen activator inhibitor-1 is influenced by very low-density lipoproteins, the expression of both tissue plasminogen activator and plasminogen activator inhibitor-1 is influenced by low-density lipoproteins and lipoprotein(a) (Lp(a)) and the internalization of the urokinase: plasminogen activator inhibitor-1 complex occurs via the low-density lipoprotein related protein. Several clinical studies have shown correlations between fibrinolytic parameters and lipoproteins in healthy populations and in patients with dyslipidemia, but the correlation between single plasma fibrinolytic enzymes and the severity of coronary atherosclerosis is less well documented. The reduction of plasma lipids with lipid-lowering drugs also affects the concentration of fibrinolytic enzymes, although this may also be due to direct effects of the drugs on the expression of the various fibrinolytic enzymes. The reduction of fibrinolytic and proteolytic activity in the atherosclerotic plaque by their lipid-lowering effect and by their direct action on the fibrinolytic system may be one of the mechanisms by which some lipid-lowering drugs achieve plaque stabilization.

Thymosin β4 induces the synthesis of plasminogen activator inhibitor 1 in cultured endothelial cells and increases its extracellular expression

AbstractThymosin β4(Tβ4), a 4.9-kDa polypeptide primarily known as a main G-actin–sequestering peptide, is present in high concentrations in various cells and in the circulation. We have found that Tβ4 upregulates the expression of plasminogen activator inhibitor 1 (PAI-1) in endothelial cells measured both at the level of mRNA and protein synthesis. This effect seems to be cell specific and was not observed when other cells such as human fibroblasts, PC3, and U937 were tested. Tβ4 significantly activated the PAI-1 promoter in EA.hy 926 cells transiently transfected either with plasmid p800LUC containing PAI-1 promoter fragment (–800 to +71) or the PAI-1 promoter linked with green fluorescent protein. Tβ4 mediated up-regulation of PAI-1 involved activation of the mitogen-activated protein kinase cascade. Furthermore, Tβ4 enhanced c-Fos/c-Jun DNA-binding activity to the activator protein 1 (AP-1)–like element (–59 to –52). The specificity of this binding activity was demonstrated by competition electrophoretic mobility shift assay and after transfection of EA.hy 926 cells with the mutated PAI-1 promoter. Taken together, these data indicate that, in response to Tβ4 stimulation, AP-1 activity increases to enhance PAI-1 transcription through its unique AP-1–like element at –59 to –52 in the PAI-1 promoter.

Plasminogen activator inhibitor-1 promoter activity in adipocytes is not influenced by the 4 G/5 G promoter polymorphism and is regulated by a USF-1/2 binding site immediately preceding the polymorphic region.

Plasminogen activator inhibitor-1 (PAI-1) levels were found to be associated with obesity indicating that adipocytes influence PAI-1 plasma levels. In addition, the 4 G/5 G promoter polymorphism of the PAI-1 gene may modulate PAI-1 transcription. We investigated the transcriptional regulation of the human PAI-1 gene in adipocytes and analyzed the genetic contribution of the 4 G/5 G polymorphism. The PAI-1 promoter was analyzed using electrophoretic mobility shift assays (EMSAs) and luciferase reporter gene assays. A putative binding site for the upstream stimulatory factor-1/2 (USF-1/2) at the polymorphic region of the PAI-1 promoter was identified. The binding of USF-1/2 was studied using nuclear extracts prepared from adipocytes and was similar in all the promoter variants as analyzed by EMSA. A 257 bp PAI-1 promoter fragment including the 4 G/5 G site was transcriptionally active in adipocytes and was not influenced by the polymorphism. The present data indicate for the first time that USF-1/2 is transcriptionally active in differentiated adipocytes. However, USF-1/2 binding activity and PAI-1 transcription are not influenced by the 4 G/5 G-allele. These data possibly explain the observation that PAI-1 secretion from adipose tissue is not influenced by the PAI-1 promoter polymorphism.

4G/5G polymorphism of the plasminogen activator inhibitor-1 gene and risk of restenosis after coronary artery stenting

Background Plasminogen activator inhibitor-1 (PAI-1) has been proposed as a candidate risk factor for restenosis after coronary artery stenting. Transcription, level, and activity of PAI-1 are influenced by the 4G/5G polymorphism in the promoter region of PAI-1 gene. The polymorphism may therefore affect wound-healing processes in injured blood vessels and influence restenosis. Methods In 1850 consecutive patients, angiographic measures of restenosis and the clinical outcome at 30 days and 1 year after stent implantation were evaluated. Angiographic restenosis was defined as ≥50% diameter stenosis determined at follow-up angiography, performed 6 months after stenting. The 4G/5G genotypes were determined with TaqMan technique. Results Among the patients, the frequency of the 4G allele was 0.55. Follow-up angiography was done in 84% of the patients. We observed restenosis in 32.5% of 4G/4G carriers, 32.2% of 4G/5G carriers, and 35.7% of 5G/5G carriers ( P = .52). The occurrence of a major adverse event (death, myocardial infarction, or target vessel revascularization due to restenosis-induced ischemia) was 5.6% in 4G/4G carriers, 5.3% in 4G/5G carriers, and 4.6% in 5G/5G carriers at 30 days ( P = .80), and 24.7% in 4G/4G carriers, 23.0% in 4G/5G carriers, and 26.2% in 5G/5G carriers at 1 year ( P = .45). Conclusion The 4G/5G polymorphism of the PAI-1 gene is not associated with an increased risk of thrombotic and restenotic events after coronary artery stenting.

4G/5G promoter polymorphism of plasminogen activator inhibitor-1, lipid profiles, and ischemic stroke

The 4G allele of common 4G/5G polymorphism in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene is associated with increased PAI-1 transcription and has been proposed as a candidate genetic risk factor for thrombotic diseases. We investigated the relationship between this polymorphism and lipid profiles and stroke risk. One hundred patients with ischemic stroke and 150 age- and sex-matched control subjects were enrolled. PAI-1 genotype was determined with the use of polymerase chain reaction and restriction-length analysis. Genotype distribution in the stroke group was 40% 4G/4G, 46% 4G/5G, and 14% 5G/5G; in the control group it was 38.7% 4G/4G, 45.3% 4G/5G, and 16% 5G/5G. The allele and genotype frequencies of 4G/5G polymorphism were not different between the stroke and control groups. Control subjects who were homozygous for the 4G allele had significantly lower high-density lipoprotein (HDL) cholesterol levels than did those carrying the 5G allele (51.2 ± 11.8 vs 58.4 ± 15.8 mg/dL; P = .002). In the control group, regression analysis revealed a significant contribution of 4G/4G genotype to increased triglyceride ( P = .042) and to decreased HDL cholesterol ( P < .001) levels. Our findings suggest that PAI-1 4G/5G promoter polymorphism alone is not associated with ischemic stroke. However, this polymorphism influences lipid levels, and the underlying mechanism must be determined.