Plasminogen Activator Inhibitor-1 Protein Expression Research Articles

Adrenomedullin inhibits angiotensin II-induced expression of tissue factor and plasminogen activator inhibitor-1 in cultured rat aortic endothelial cells.

Adrenomedullin (AM) is a potent vasodilating peptide having a variety of pharmacological properties mainly in respect to vascular pathophysiology. We have previously demonstrated that angiotensin II (Ang II) or natriuretic peptides have influence on the expression of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) in vascular endothelial cells. The aim of this study was to elucidate the effects of AM on TF and PAI-1 mRNA and protein expression in endothelial cells. As a result, AM inhibited Ang II-induced TF and PAI-1 mRNA expression in a dose- and time-dependent manner. Because the expression of TF and PAI-1 mRNA induced by Ang II was attenuated by the increase of intracellular concentrations of cAMP by forskolin and 8-bromo-cAMP and because AM increased the intracellular level of cAMP in rat aortic endothelial cells, it was indicated that the inhibitory effect of AM on the expressions of TF and PAI-1 was mainly mediated by the cAMP-dependent signal transduction. Furthermore, the inhibitory effect of AM on TF and PAI-1 expression was partly attenuated by an NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester. In conclusion, AM is shown to contribute to the regulation of blood coagulation and fibrinolysis by vascular endothelial cells mainly via the cAMP pathway.

Plasminogen activator inhibitor-1 and its cofactor vitronectin stabilize arterial thrombi after vascular injury in mice.

The origin and contribution of plasminogen activator inhibitor-1 (PAI-1) and its cofactor vitronectin (VN) to arterial thrombosis/thrombolysis in vivo is controversial. Ferric chloride was used to induce carotid artery injury in 97 wild-type (WT), 84 PAI-1-/-, and 84 VN-/- mice. Complete thrombotic occlusion was observed in 70% of PAI-1-/- mice versus 92% of WT (P:<0.001) and 87% of VN-/- (P:=0.015) mice. In vessels that occluded, mean times to occlusion were significantly longer in PAI-1-/- than in WT or VN-/- mice. The initial thrombotic response of VN-/- mice was similar to that of WT mice, but their thrombi were unstable and frequently embolized. As a result, the patency rate of carotid vessels 30 minutes after injury was as high in VN-/- mice (36%) as in PAI-1-/- mice (which demonstrate progressive thrombolysis) and significantly higher than that of WT mice (12%; P:=0.013). Histochemical and reverse transcription-polymerase chain reaction studies revealed an early upregulation of PAI-1 mRNA and protein expression in the thrombus and the vessel wall, which persisted for >/=1 week. VN protein also accumulated after injury, but VN mRNA levels remained low at all times. PAI-1 and VN participate in the thrombotic response to arterial injury by preventing premature thrombus dissolution and embolization. The accumulation of PAI-1 in the thrombus/vessel wall after injury may result, at least in part, from local synthesis, whereas the VN protein appears to be derived from plasma.

Effect of nitric oxide modulation on TGF-β1 and matrix proteins in chronic cyclosporine nephrotoxicity

Chronic cyclosporine (CsA) nephrotoxicity is characterized by interstitial fibrosis and afferent arteriolar hyalinosis. L-arginine (L-Arg), the substrate for nitric oxide (NO) synthase and N-nitro-L-arginine-methyl ester (L-NAME), the NO synthase inhibitor, were shown to modulate acute CsA nephrotoxicity. However, the mechanism of fibrosis in chronic CsA nephrotoxicity remains unclear. Thus, we examined the effect of NO modulation on fibrosis and the expression of transforming growth factor-beta1 (TGF-beta1) and matrix proteins in chronic CsA nephrotoxicity. Rats were administered CsA (7.5 mg/kg), CsA + L-Arg (1.7 g/kg), CsA + L-NAME (3.5 mg/kg), vehicle (VH), VH + L-Arg, and VH + L-NAME, and were sacrificed at 7 or 28 days. NO production, physiologic parameters, and histology were studied in addition to the mRNA expression of TGF-beta1, plasminogen activator inhibitor-1 (PAI-1) and the matrix proteins biglycan and collagens type I and IV by Northern and the protein expression of PAI-1 and fibronectin by enzyme-linked immunosorbent assay. While L-NAME strikingly reduced NO biosynthesis and worsened the glomerular filtration rate and CsA-induced fibrosis, L-Arg had the opposite beneficial effect. In addition, the CsA-induced up-regulated expression of TGF-beta1, PAI-1, and the matrix proteins biglycan, fibronectin, and collagen I was significantly increased with L-NAME and strikingly improved with L-Arg. Collagen IV expression was not affected. Also, NO modulation did not affect VH-treated rats. Chronic CsA nephrotoxicity can be aggravated by NO blockade and ameliorated by NO enhancement, suggesting that NO maintains a protective function. NO modulation was associated with a change in TGF-beta1 expression, which, in turn, was associated with alterations in matrix deposition and matrix degradation through its effect on PAI-1.

In Vivo Stimulation of Vascular Plasminogen Activator Inhibitor-1 Production by very Low-Density Lipoprotein Involves Transcription Factor Binding to a VLDL-Responsive Element

High plasma levels of plasminogen activator inhibitor-1 (PAI-1) are associated with an increased risk of cardiovascular disease. There is also a close relation between high plasma levels of PAI-1 and hypertriglyceridemia. Cell culture studies have shown that very low density lipoprotein (VLDL) increases the production and secretion of PAI-1 in endothelial cells and hepatocytes, suggesting a possible mechanism for this association. To determine whether VLDL stimulates PAI-1 production in vascular cells also in vivo, Sprague-Dawley rats were injected intravenously with 6 mg/kg of VLDL (derived from human subjects with type IV hyperlipidemia). Previous studies have demonstrated that this results in an accumulation of human VLDL in the aorta and other arteries followed by increased nuclear factor-kappa B (NF-kappaB) activation. Endothelial, but not smooth muscle cells, showed a basal PAI-1 mRNA and protein expression as assessed by in situ hybridization and immunohistochemistry, respectively. Six to twenty-four hours after the VLDL injection, lipoprotein particle accumulation was seen in the aortic wall, which was accompanied by increasing PAI-1 mRNA and protein expression in endothelial and smooth muscle cells. Within the rat PAI-1 promoter we identified a sequence located at -589 to -571 with 74% homology with the recently described VLDL responsive element in the human PAI-1 promoter and located adjacent to a 4-guanosine motif presumably corresponding to the human 4G/5G polymorphism. Transient transfection studies showed that VLDL exerts its stimulatory effects on rat PAI-1 gene expression in vascular cells by interaction with promoter sequences located within bp -656 and -505. Electrophoretic mobility shift assays showed that VLDL increases the binding of as yet incompletely characterized factors to this response element. Taken together these observations support a direct influence of VLDL on vascular PAI-1 gene expression ill vivo. This stimulation is exerted on the level of PAI-1 gene transcription, and involves transcription factor binding to a VLDL responsive element adjacent to a 4G motif within the PAI-1 promoter.

Oviductal plasminogen activator inhibitor-1 (PAI-1): mRNA, protein, and hormonal regulation during the estrous cycle and early pregnancy in the pig.

Recent identification of plasminogen activator inhibitor-1 (PAI-1) in the pig oviduct has prompted an evaluation of its mRNA, protein synthesis, and hormonal regulation during the estrous cycle and early pregnancy, defined as time prior to and after maternal recognition of pregnancy. To examine PAI-1 protein synthesis, oviductal tissue was collected from European Large White and Chinese Meishan gilts on days 0, 2, and 5 of early pregnancy, divided into three functional segments, and cultured. Culture media was collected and de novo synthesized PAI-1 analyzed by 2D-SDS-PAGE, fluorography, and densitometry. To determine hormonal regulation of PAI-1 synthesis and secretion, four groups of ovariectomized (OVX) cross-bred gilts were each treated with one of four steroid regimens (corn oil, estrogen, progesterone, or estrogen + progesterone) and tissue collected for RNA or cultured. Steady-state mRNA levels of PAI-1 were evaluated throughout the estrous cycle in cross-bred gilts. To compare steady-state PAI-1 mRNA levels between cyclic and pregnant cross-bred gilts, tissue was collected on days 0, 2, and 12. Quantitative analysis of steady-state levels of PAI-1 mRNA were analyzed by dot-blot hybridization and densitometry. A greater (P < 0.01) synthesis and secretion of PAI-1 protein was found in the isthmus portion of the oviduct relative to either the ampulla or infundibulum regardless of day of pregnancy or breed. No difference could be detected for PAI-1 protein between breeds. The Large White had a greater (P < 0.05) secretion of PAI-1 on day 2 of early pregnancy relative to other days examined. Whole oviductal tissue from cross-bred gilts was found to have a significantly greater amount of PAI-1 mRNA on days 1 and 2 compared to other days examined, while the isthmus had significantly greater levels of mRNA on days 2 and 12. A significant effect of day and segment was detected for levels of PAI-1 mRNA from cyclic and early pregnant cross-bred gilts. PAI-1 mRNA was found to be significantly greater in the isthmus than other segments, regardless of day of the estrous cycle or pregnancy. An interaction was detected for estrogen and progesterone on PAI-1 mRNA (P < 0.05) and protein (P = 0.09). Estrogen was found to inhibit PAI-1 protein synthesis and also inhibited progesterone-mediated stimulation of PAI-1 mRNA. Our results demonstrate expression of PAI-1 mRNA and protein are highest on day 2 of early pregnancy, which is consistent with its proposed function of protecting the oocyte/embryo from enzymatic degradation and/or extracellular matrix remodeling of both oviduct and early cleavage-stage embryo.

Induction of the Plasminogen Activator Inhibitor-1 Gene Expression by Mild Hypoxia Via a Hypoxia Response Element Binding the Hypoxia-Inducible Factor-1 in Rat Hepatocytes

Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of both tissue-type and urokinase-type plasminogen activators. The balance between plasminogen activators and PAI-1 plays an important role in several physiological and pathophysiological processes such as atherosclerosis or thrombosis. Because these conditions are associated with hypoxia, it was the aim of the present study to investigate the influence of low O2tension on the expression of PAI-1 mRNA and protein using primary cultured rat hepatocytes as a model system. We found that PAI-1 mRNA and protein were induced by mild hypoxia (8% O2). The hypoxia-dependent PAI-1 mRNA induction was transcriptionally regulated because it was inhibited by actinomycin D (ActD). Luciferase (LUC) reporter gene constructs driven by about 800 bp of the 5′-flanking region of the rat PAI-1 gene were transiently transfected into primary rat hepatocytes; mild hypoxia caused a 3-fold induction, which was mediated by the PAI-1 promoter region -175/-158 containing 2 putative hypoxia response elements (HRE) binding the hypoxia-inducible factor (HIF-1). Mutation of the HRE-1 (-175/-168) or HRE-2 (-165/-158) also abolished the induction by mild hypoxia. Cotransfection of a HIF-1 vector and the PAI-1–LUC constructs, as well as gel shift assays, showed that the HRE-2 of the PAI-1 promoter was most critical for induction by hypoxia and HIF-1 binding. Thus, PAI-1 induction by mild hypoxia via a HIF-1 binding HRE in the rat PAI-1 promoter appears to be the mechanism causing the increase in PAI-1 in many clinical conditions associated with O2deficiency.

Increased expression of plasminogen activator and plasminogen activator inhibitor during liver fibrogenesis of rats: role of stellate cells

Background/Aims: Plasminogen activators and plasminogen activator inhibitors are important regulators of the balance between the proteolytic and antiproteolytic activities that determine extracellular matrix turnover. We examined the expression of plasminogen activator-plasmin system components in experimental liver fibrosis of rats.Methods: Liver fibrosis was produced in rats by injecting carbon tetrachloride for 6 to 12 weeks. Gene expression for plasminogen activator inhibitor-1 (PAI-1), urokinase and tissue plasminogen activators (uPA and tPA), urokinase plasminogen activator receptor (uPAR), and transforming growth factor-β1 (TGF-β1) was examined by Northern analysis. Western analysis was performed to detect protein expression of PAI-1, uPA and uPAR. An immunohistochemical study was performed to detect the localization of PAI-1. Additionally, primary cultured liver cells were examined by Northern and Western analyses for this protein with or without prior incubation with TGF-β1.Results: At 6 weeks, when fibrosis had occurred, uPA and uPAR mRNAs had increased 2.8-fold and 1.8-fold, respectively; PAI-1 and tPA mRNA levels were unchanged. At the cirrhotic stage (9 to 12 weeks), mRNA levels for PAI-1, uPA, uPAR and tPA were all increased. Western analysis also showed increased uPA and uPAR expressions in fibrotic liver, and increased PAI-1, uPA and uPAR expressions in cirrhotic liver. PAI-1 protein was also demonstrated immunohistochemically along sinusoids, vessels, and bile duct cells of normal and fibrotic liver. In liver cell cultures, Kupffer cells, hepatocytes, and especially stellate cells, expressed PAI-1. Expression was enhanced in stellate cells cultured from fibrotic or cirrhotic liver or stimulated in vitro with TGF-β1.Conclusion: Though increased uPA and uPAR may act on matrix degradation in fibrotic liver, increased PAI-1 together with uPA, uPAR and tPA are associated with overall inhibition of matrix degradation in cirrhotic liver. Hepatic stellate cells are an important source of PAI-1 during liver fibrosis.

Angiotensin IV stimulates plasminogen activator inhibitor-1 expression in proximal tubular epithelial cells

Angiotensin II (Ang II) has been shown to be implicated in the development of renal fibrosis in several forms of chronic glomerulonephritides, but the precise mechanisms of its effects remain unclear. It has recently been reported that Ang II stimulates the expression of plasminogen activator inhibitor-1 (PAI-1) in several cell lines. PAI-1 is a major physiological inhibitor of the plasminogen activator/plasmin system, a key regulator of fibrinolysis and extracellular matrix (ECM) turnover. PAI-1 induction by Ang II in endothelial cells seems to be mediated by Ang IV via a receptor that is different from Ang II type 1 and 2 receptors (AT1 and AT2). In this study, we sought to evaluate the effects of Ang IV on PAI-1 gene and protein expression in a well-characterized and immortalized human proximal tubular cell line (HK2) by Northern blot and enzyme-linked immunosorbent assay. Ang IV stimulated PAI-1 mRNA expression, whereas it did not induce a significant increase in tritiated thymidine uptake after 24 hours of incubation. This effect was dose and time dependent. Ang IV (10 nM) induced a 7.8 +/- 3.3-fold increase in PAI-1 mRNA expression. The PAI-1 antigen level was significantly higher in conditioned media and the ECM of cells treated with Ang II and Ang IV than in control cells (both P < 0.02). Although Ang II induced a 4.2 +/- 2. 1-fold increase in PAI-1 mRNA expression, its effect underwent a dose-dependent reduction when amastatin, a potent inhibitor of the endopeptidases that catalyzes the conversion of Ang II to Ang IV, was added. In contrast, amastatin was not able to prevent the expression of PAI-1 mRNA induced by Ang IV. Finally, pretreatment of HK2 cells with losartan and N-Nicotinoyl-Tyr-N3-(Nalpha-CBZ-Arg)-Lys-His-Pro-Ile, the specific antagonists of AT1 and AT2 receptors, failed to modify PAI-1 mRNA expression as induced by Ang II. Our results demonstrate that Ang II stimulates PAI-1 mRNA expression and the production of its protein in human proximal tubular cells. This is mainly-if not exclusively-due to Ang IV, which acts on a receptor that is different than AT1 or AT2. Therefore, it can be hypothesized that the induction of PAI-1 by Ang IV may be implicated in the pathogenesis of renal interstitial fibrosis in several forms of chronic glomerulonephritides.

Expression of urokinase-type plasminogen activator receptor and plasminogen activator inhibitor-1 in gastric cancer.

In gastric cancer, the urokinase-type plasminogen activator (uPA) system plays important roles in invasion and metastasis, processes which entail proteolysis and adhesion. Both the urokinase-type plasminogen activator receptor (uPAR) and the plasminogen activator inhibitor-1 (PAI-1) are thought to be important factors in this system. To clarify the relationship between these two factors and gastric cancer invasiveness, we evaluated the expression of uPAR and PAI-1 in 91 cases of gastric cancer by immunohistochemistry and in situ hybridization. Urokinase-type plasminogen activator receptor-mRNA, PAI-1-mRNA, uPAR and PAI-1 protein were diffusely distributed in the cytoplasm of the cancer cells and concentrated at invasive foci. Urokinase-type plasminogen activator receptor protein expression correlated with lymphatic, venous invasion (P< 0.01) and lymph node metastasis (P< 0.05); uPAR-mRNA expression correlated with lymphatic, venous invasion and lymph node metastasis (P< 0.05). Plasminogen activator inhibitor-1 protein expression correlated with lymphatic, venous invasion, lymph node metastasis and depth of invasion (P<0.01); PAI-1-mRNA expression was linked to lymphatic, venous invasion (P< 0.01), lymph node metastasis and depth of invasion (P< 0.05). This suggests that the proteolytic activity of uPAR and the cellular motility of PAI-1 in gastric cancer cells may determine penetration of lymphatic and blood vessels, whereby lymph node metastasis may be promoted and that the promotion of cellular motility by PAI-1 may influence the depth of cancer invasion.

Plasminogen activator inhibitor-1 expression by brain microvessel endothelial cells is inhibited by elevated glucose.

Patients with diabetes are predisposed to microvascular disease. In the retina and brain, this is characterized by neovascularization and new capillary formation. Because of the potential importance of plasmin generation in these processes, we evaluated the effect of elevated glucose concentrations on expression of plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA), and urokinase (uPA) in cultured bovine brain endothelial cells (BBEC) versus cultured bovine aortic endothelial cells (BAEC). We observed that BBEC PAI-1 mRNA levels were decreased fivefold in cells cultured in media containing 20 mM glucose compared with BBEC cultured in media with 5.5 mM glucose, whereas expression of PAI-1 mRNA in BAEC, bovine mesenteric endothelial cells, and human umbilical vein endothelial cells was not modulated under these conditions. Expression of PAI-1 protein was also inhibited by growth of BBEC in elevated glucose, but the effect was less marked than at the mRNA level. Elevated glucose did not decrease expression of PAI-1 protein by BAEC. Withdrawal of acidic fibroblast growth factor enhanced expression of PAI-1 mRNA and protein in BBEC. Expression of tPA mRNA was not affected by the glucose concentration of the medium, and uPA mRNA was not detected in our BBEC cultures. A decrease in the local tissue activity of PAI-1 by elevated glucose concentrations, with no effect on tPA or uPA expression, would lead to an increase in the plasmin activity and thereby predispose neural tissues, such as the cerebrum and retina, of diabetic patients to neovascularization.