Regulation of plasminogen activator inhibitor-1 secretion by growth factors in smooth muscle cells.

Epithelioid-type vascular smooth muscle cells are metabolically active and secrete many proteases and protease inhibitors. We have previously cloned epithelioid-type smooth muscle cells from rat carotid arteries, and showed that polypeptide growth factors basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) could dose-dependently induce plasminogen activator inhibitor-1 (PAI-1) secretion from these cells. In the present study, we have used these cells to investigate the growth factor-induced signal transduction pathways leading to PAI-1 secretion. We report here that PAI-1 induction was dependent on protein kinase C (PKC) and tyrosine kinase but not on protein kinase A (PKA), ras and phosphoinositol-3-kinase inhibitor. Induction of PAI-1 by bFGF and PDGF was also accompanied by activation of a mitogen-activated protein kinase pathway involving Raf/Mek/Erk1/2, and the family non-receptor tyrosine kinases., another non-receptor tyrosine kinase, on the contrary, behaved differently from in that it was part of a pathway leading to PAI-1 induction by bFGF, but not when PDGF was used as the stimulating reagent. Activation of a PKA-dependent pathway(s) opposed PAI-1 induction. One mechanism for PKA activators to inhibit PAI-1 secretion was that they markedly inhibited the phosphorylations of Mek and mitogen-activated protein kinase that were up-regulated in the presence of bFGF and PDGF.     

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-beta1 (TGF-beta1) 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-beta1. 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-beta1. 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.     

Ovulation efficiency is reduced in mice that lack plasminogen activator gene function: functional redundancy among physiological plasminogen activators.

Several lines of indirect evidence suggest that plasminogen activation plays a crucial role in degradation of the follicular wall during ovulation. However, single-deficient mice lacking tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), or PA inhibitor type 1(PAI-1) gene function were recently found to have normal reproduction, although mice with a combined deficiency of tPA and uPA were significantly less fertile. To investigate whether the reduced fertility of mice lacking PA gene function is due to a reduced ovulation mechanism, we have determined the ovulation efficiency in 25-day-old mice during gonadotropin-induced ovulation. Our results reveal that ovulation efficiency is normal in mice with a single deficiency of tPA or uPA but reduced by 26% in mice lacking both physiological PAs. This result suggests that plasminogen activation plays a role in ovulatory response, although neither tPA nor uPA individually or in combination is obligatory for ovulation. The loss of an individual PA seems to be functionally complemented by the remaining PA but this compensation does not appear to involve any compensatory up-regulation. Our data imply that a functionally redundant mechanism for plasmin formation operates during gonadotropin-induced ovulation and that PAs together with other proteases generate the proteolytic activity required for follicular wall degradation.     

Urokinase-type plasminogen activator receptor regulates leukocyte recruitment during experimental pneumococcal meningitis

Tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) have been suggested to play an important role in inflammatory diseases. Increased levels of tPA, uPA, uPA receptor (uPAR), and their inhibitor, plasminogen activator inhibitor (PAI)-1, have been found in the cerebrospinal fluid (CSF) of patients with bacterial meningitis. Here, we show that expression of tPA, uPA, uPAR, PAI-1, and PAI-2 is up-regulated during experimental pneumococcal meningitis. In uPAR-deficient mice, CSF pleocytosis was significantly attenuated 24 h after infection, compared with that in infected wild-type (wt) mice. Lack of uPAR did not influence blood-brain barrier permeability, intracranial pressure, expression of chemokines (keratinocyte-derived cytokine and macrophage inflammatory protein-2), bacterial killing, or clinical outcome. No differences in pathophysiological alterations were observed in tPA-deficient mice, compared with those in infected wt mice. These results indicate that uPAR participates in the recruitment of leukocytes to the CSF space during pneumoccal meningitis.     

Very low density lipoprotein-mediated signal transduction and plasminogen activator inhibitor type 1 in cultured HepG2 cells.

In normal subjects and in patients with cardiovascular disease, plasma triglycerides are positively correlated with plasminogen activator inhibitor type 1 (PAI-1) levels. Moreover, in vitro studies indicate that VLDLs induce PAI-1 synthesis in cultured cells, ie, endothelial and HepG2 cells. However, the signaling pathways involved in the effect of VLDL on PAI-1 synthesis have not yet been investigated. We report that VLDLs induce a signaling cascade that leads to an enhanced secretion of PAI-1 by HepG2 cells. In myo-[(3)H]inositol-labeled HepG2 cells, VLDL (100 microg/mL) caused a time-dependent increase in [(3)H]inositol phosphates, the temporal sequence being tris>bis>monophosphate. VLDL brought about a time-dependent stimulation of membrane-associated protein kinase C (PKC) activity and arachidonate release. Finally, VLDL stimulated mitogen-activated protein (MAP) kinase, and this effect was reduced by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), which suggests that PKC plays a pivotal role in MAP kinase phosphorylation. VLDL-induced PAI-1 secretion was completely prevented by U73122, a specific inhibitor of phosphatidylinositol-specific phospholipase C, by H7 or by PKC downregulation, and by mepacrine (all P<0.01 versus VLDL-treated cells). 3,4,5-Trimethoxybenzoic acid 8-(diethylamino)-octyl ester, which prevents Ca2+ release from intracellular stores, inhibited VLDL-induced PAI-1 secretion by 60% (P<0.05), and the MAP kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor PD98059 completely suppressed both basal and VLDL-induced PAI-1 secretion. These data demonstrate that VLDL-induced PAI-1 biosynthesis results from a principal signaling pathway involving PKC-mediated MAP kinase activation.     

Hypofibrinolysis in patients with hypercoagulability: The roles of urokinase and of plasminogen activator inhibitor

The prevalence of abnormalities of fibrinolysis in patients with venous thromboembolism is as yet unknown. Defined abnormalities include congenital dysfunction and deficiency of plasminogen, and probably impaired plasminogen activation secondary to elevated levels of plasminogen activator inhibitor type 1 (PAI-1) or to impaired release of tissue plasminogen activator (tPA). In this preliminary study, we analyzed plasma samples from 21 patients for whom an investigation for possible thrombophilia was requested. Twenty of the patients had venous thromboembolism, and one had arterial thrombosis at an early age. Two patients had deficiency of protein C or protein S, but no other recognized biochemical disturbances related to thrombophilia were identified. Patient samples and plasma from 25 normal controls were assayed for tPA activity, PAI-1 activity, and urokinase (uPA) activity and antigen. tPA activity and antigen were not significantly different in patients than in controls. PAI-1 activity was significantly greater in patients (P < 0.0001). uPA activity was not different in the two groups. However, uPA antigen was significantly reduced in patients compared to controls (P = 0.001). These data suggest that hypofibrinolysis leading to a risk of thrombosis may be caused not only by elevated PAI-1 activity but also by reduced total uPA concentration. © 1993 Wiley-Liss, Inc.     

纤维蛋白原、纤维蛋白及其降解产物对共培养血管内皮细胞纤溶活性的影响

目的研究纤维蛋白原（Fg）、纤维蛋白（Fb）及其降解产物（FDP）对共培养体系中人脐静脉内皮细胞组织型纤溶酶原激活物和纤溶酶原激活物抑制剂表达的影响。方法应用Transwell膜建立人脐静脉内皮细胞-兔主动脉平滑肌细胞共培养体系，在不同浓度（0、0．5、1．5、3．0、4．5和6．0g／L）Fg、Fb和FDP干预24h后，分别检测该共培养体系中人脐静脉内皮细胞组织型纤溶酶原激活物和纤溶酶原激活物抑制剂mRNA水平（RT-PCR法）以及培养上清中组织型纤溶酶原激活物和纤溶酶原激活物抑制剂抗原含量（ELISA法）与活性（发色底物法）的变化情况。结果Fg对组织型纤溶酶原激活物的表达没有显著影响，较高浓度的Fg（3．0～4．5g／L）可明显促进纤溶酶原激活物抑制剂mRNA表达、抗原含量及活性升高，但过高浓度的Fg（6．0g／L）却抑制纤溶酶原激活物抑制剂的表达。3．0-4．5g／L的Fb对组织型纤溶酶原激活物mRNA和抗原含量都起上调作用，同时显著下调组织型纤溶酶原激活物活性。较高浓度的Fb（1．5-4．5g／L）则可明显上调纤溶酶原激活物抑制剂的表达，且在mRNA、蛋白和活性水平趋势基本一致。3．0-6．0g／L的FDP均可明显下调组织型纤溶酶原激活物mRNA、蛋白和活性水平，1．5-6．0mg／ml的FDP均可促进纤溶酶原激活物抑制剂的高表达。结论Fg、Fb和FDP可以通过影响组织型纤溶酶原激活物和纤溶酶原激活物抑制剂的表达，引起纤溶活性降低，参与动脉粥样硬化的发展进程。     

Fibronectin-induced proliferation in thyroid cells is mediated by alphavbeta3 integrin through Ras/Raf-1/MEK/ERK and calcium/CaMKII signals

We recently demonstrated in an immortalized thyroid cell line that integrin stimulation by fibronectin (FN) simultaneously activates two signaling pathways: Ras/Raf/MAPK kinase (Mek)/Erk and calcium Ca2+/calcium calmodulin-dependent kinase II (CaMKII). Both signals are necessary to stimulate Erk phosphorylation because CaMKII modulates Ras-induced Raf-1 activity. In this study we present evidence that extends these findings to normal human thyroid cells in primary culture, demonstrating its biological significance in a more physiological cell model. In normal thyroid cells, immobilized FN-induced activation of p21Ras and Erk phosphorylation. This pathway was responsible for FN-induced cell proliferation. Concurrent increase of intracellular Ca2+ concentration and CaMKII activation was observed. Both induction of p21Ras activity and increase of intracellular Ca2+ concentration were mediated by FN binding to alphavbeta3 integrin. Inhibition of the Ca2+/CaMKII signal pathway by calmodulin or CaMKII inhibitors completely abolished the FN-induced Erk phosphorylation. Binding to FN induced Raf-1 and CaMKII to form a protein complex, indicating that intersection between Ras/Raf/Mek/Erk and Ca2+/CaMKII signaling pathways occurred at Raf-1 level. Interruption of the Ca2+/CaMKII signal pathway arrested cell proliferation induced by FN. We also analyzed thyroid tumor cell lines that displayed concomitant aberrant integrin expression and signal transduction. These data confirm that integrin activation by FN in normal thyroid cells generates Ras/Raf/Mek/Erk and Ca2+/CaMKII signaling pathways and that both are necessary to stimulate cell proliferation, whereas in thyroid tumors integrin signaling is altered.     

Regulation of endothelial tissue plasminogen activator and plasminogen activator inhibitor type 1 synthesis by diacylglycerol,phorbol ester,and thrombin

Summary The influence of diacylglycerols, which are physiological activators of protein kinase C, on the production of tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAI-1) by human umbilical vein endothelial cells (HUVEC) was studied in order to gain insight into the regulation of fibrinolysis by these cells. 1,2-dioctanoyl-sn-glycerol (diC₈) stimulated tPA production in a dose- and time-dependent manner. The tPA antigen in cell supernatants increased from 0.9 ng/10⁶ cells in unstimulated cells to 12.4 ng (10⁶ cells after incubation with 400µM diC₈ for 24 hours. In contrast, PAI-1 production was not influenced by diC₈, whereas phorbol 12-myristrate 13-acetate (PMA) or thrombin stimulated both, tPA and PAI-1 production by HUVEC. Staurosporine and H7, which are inhibitors of protein kinase C, inhibited tPA synthesis by HUVEC. The degree of inhibition was dependent on the agonist used. While diC₈-induced tPA production was inhibited to more than 80% by H7 (10µM) and staurosporine (10 nM), higher doses of inhibitors were required to inhibit thrombin- and PMA-induced tPA production. Thrombin-induced PAI-1 production was inhibited to more than 80% by H7 (10µM) and to about 50% by staurosporine, whereas PMA-induced PAI-1 production was not inhibited by staurosporine, and only to about 50% by higher doses of H7 (30µM). These data suggest that activation of protein kinase C is a common intracellular trigger mechanism for the induction of tPA synthesis by HUVEC. Protein kinase C is most likely also involved in the regulation of PAI-1 synthesis by HUVEC.     

Protection by α2-macroglobulin of tissue plasminogen activator against inhibition by plasminogen activator inhibitor-1

Tissue plasminogen activator (tPA) is widely used in the treatment of acute myocardial infarction (MI). However, its thrombolytic efficacy does not correlate with the dose administered. The interactions between tPA, α2-macroglobulin (α2-M), and plasminogen activator inhibitor-1 (PAI-1) were investigated both in vitro and in patients undergoing tPA therapy for MI in an attempt to identify variables that might affect the clinical efficacy of tPA.
Purified α2-M (5.4 mg/ml) protected 16.0% or 22.4% of tPA (12.5 IU/ml) activity from inhibition by PAI-1 at 4 or 8 IU/ml in vitro . Of nine patients treated with 5–20 mega IU of tPA for MI, the plasma activity of tPA remained increased for 15–30 min after the cessation of infusion in eight; the patient who failed to exhibit a persistent increase in tPA activity had a low plasma concentration of α2-M. Total tPA activity, derived from the area under the activity-versus-time curve (AUC), showed a significant inverse correlation with the ratio of the plasma PAI-1 activity to the plasma α2-M concentration. Total tPA activity did not correlate with plasma PAI-1 activity or plasma α2-M concentration alone. Results suggest that α2-M, by binding to tPA, protects the latter against inhibition by PAI-1.     

Stimulation of plasminogen activator and inhibitor in the lymphatic endothelium

Chromogenic assays, immunoblotting, and Northern blot hybridization methods were employed to assess the effects of various agonists on the production of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) by the lymphatic endothelium (LEC). Fibrin autography showed that plasminogen-dependent fibrinolytic activity occurred at M(r) of 110 kDa, which represents a complex of tPA with PAI-1, and 65- and 55-kDa bands corresponding to tPA and uPA, respectively. The fractionation of lymph collected from ovine lymphatic vessels also produced a prominent lytic band of approximately 110 kDa, suggesting the formation of PA/PAI complexes in lymph. The stimulation of various agonists produced large-scale increases in tPA mRNA, as shown by Northern blot hybridization analyses. The effects of ECGF, histamine, and LPS on the presence of tPA and on enhancing the levels of mRNA reached maximum activity at 4 h and declined to levels below that of controls by 8 h. However, phorbol-treated cells exhibited reduced levels of tPA mRNA at 4 h, but was significantly increased by 8 h. A large-scale increase in PAI-1 mRNA steady-state levels was also stimulated by the agonists used in these studies. Both the 3.4- and 2.4-kb species of PAI-1 mRNA were increased. These observations demonstrated that tPA and PAI-1 are produced and secreted by LEC monolayer cultures and are also present in lymph.     

Beyond fibrinolysis: the role of plasminogen activator inhibitor-1 and vitronectin in vascular wound healing

Plasminogen activator inhibitor-1 (PAI-1), as the name implies, is the primary in vivo inhibitor of both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). PAI-1 also binds to other nonproteinase ligands, including the matrix protein vitronectin, glycosaminoglycans such as heparin, and the endocytic clearance receptor, the low-density-lipoprotein-receptor-related protein (LRP). PAI-1 belongs to the superfamily of serine proteinase inhibitors (serpins), and, like other serpins, it acts as "suicide inhibitor" that reacts only once with a target proteinase. The suicide mechanism results in irreversible modification of the serpin and an extensive change in its conformation. In the case of PAI-1, this conformational change is important not only for inhibition of the proteinase, but it also causes changes in affinity for vitronectin and LRP. These changes have important consequences for cell migration.     

Smooth muscle cells express urokinase during mitogenesis and tissue-type plasminogen activator during migration in injured rat carotid artery

Although the level of plasminogen activator (PA) expression has been correlated with cellular proliferation and migration in vitro, this relation has not been established in tissue undergoing repair. In a rat model of arterial injury, we have measured the expression of PAs by vascular smooth muscle cells (SMCs) during entry into the growth cycle (0-24 hours) and subsequent migration from the media to the intima (starting at approximately 4 days). In normal rat carotid, low levels of urokinase-type PA (uPA) and tissue-type PA (tPA) are present; after removal of the endothelium, only uPA is detected in the media. uPA activity in extracts of carotid arteries increases and reaches a maximum between 16 and 24 hours after injury; uPA mRNA increases steadily and is maximal at 7 days. tPA activity appears at 3 days and is maximal at 7 days; tPA mRNA is present in normal vessels and reaches a maximum by 7 days. Most of the tPA in the media is associated with SMC and not with regenerating endothelium. Furthermore, tPA is present in the media before the SMCs migrate into the intima. These results demonstrate that PA expression by vascular SMCs is differentially regulated, with uPA present during mitogenesis and tPA during migration.     

Glucocorticoid regulation of plasminogen activator inhibitor-1 messenger ribonucleic acid and protein in normal and malignant rat osteoblasts.

Glucocorticoids exert potent inhibitory effects on bone formation. We have previously shown that glucocorticoids suppress plasminogen activator (PA) activity in normal and malignant rat osteoblasts. To clarify the mechanism of this suppression, we investigated the effects of dexamethasone on PA inhibitor-1 (PAI-1), tissue-type PA (tPA), and urokinase-type PA (uPA) expression and also on PAI-1 protein and PA activity in both normal rat calvarial osteoblasts and a clonal osteogenic sarcoma cell line, UMR 106-01. Dexamethasone increased PAI-1 mRNA and protein in both cell types. The increase in PAI-1 protein and the decrease in PA activity were obtained over the same concentration range, with a half-maximally effective concentration of dexamethasone of about 10(-9) M. The increase in PAI-1 mRNA caused by dexamethasone was retained with cycloheximide treatment, but abolished with actinomycin-D. Dexamethasone had no effect on tPA or uPA mRNA in either cell type. The glucocorticoid antagonist RU 486 prevented the effects of dexamethasone on PA activity and PAI-1 protein. Dihydrotestosterone, progesterone, and 17 beta-estradiol did not influence PA activity or PAI-1 formation. Although tPA and uPA protein could not be measured, these results suggest that glucocorticoids suppress PA activity predominantly by increasing PAI-1 synthesis in rat osteoblasts. Suppression of PA activity through actions on PAI-1 formation by glucocorticoids could contribute to the mechanisms by which glucocorticoids inhibit bone formation.     

Tissue-type plasminogen activator deficiency exacerbates cholestatic liver injury in mice

Recent studies demonstrating a role for plasminogen activator inhibitor (PAI)-1 in cholestatic liver disease in mice suggested that tissue-type plasminogen activator (tPA) or urokinase plasminogen activator (uPA) might be important after biliary tract obstruction. We now demonstrate that blocking tPA exacerbates liver injury after bile duct ligation (BDL). tPA deficient mice have increased bile infarcts, increased TUNEL positive cells, increased neutrophil infiltration, reduced hepatocyte proliferation and reduced ductular reaction 72 hours after BDL compared to wild type mice. In addition, the protective and proliferative effects of plasminogen activator inhibitor 1 (PAI-1) deficiency after BDL are dramatically blocked by the tPA inhibitor tPA-STOP. One potential mechanism for these effects is that both tPA deficiency and tPA-STOP reduce hepatocyte growth factor (HGF) activation and c-Met phosphorylation in the liver after BDL. In support of this hypothesis, HGF treatment reverses the effects of tPA deficiency in mice. Furthermore, preferential tPA activation in areas of injury after BDL might occur because fibrin accumulates in bile infarcts and activates tPA. CONCLUSION: tPA inactivation accelerates liver injury after BDL and reduces HGF activation. These data suggest that strategies to increase HGF activation might be protective in liver diseases with biliary tract obstruction even without increased HGF production.