PAI-1 in tissue fibrosis

Fibrosis is defined as a fibroproliferative or abnormal fibroblast activation-related disease. Deregulation of wound healing leads to hyperactivation of fibroblasts and excessive accumulation of extracellular matrix (ECM) proteins in the wound area, the pathological manifestation of fibrosis. The accumulation of excessive levels of collagen in the ECM depends on two factors: an increased rate of collagen synthesis and or decreased rate of collagen degradation by cellular proteolytic activities. The urokinase/tissue type plasminogen activator (uPA/tPA) and plasmin play significant roles in the cellular proteolytic degradation of ECM proteins and the maintenance of tissue homeostasis. The activities of uPA/tPA/plasmin and plasmin-dependent MMPs rely mostly on the activity of a potent inhibitor of uPA/tPA, plasminogen activator inhibitor-1 (PAI-1). Under normal physiologic conditions, PAI-1 controls the activities of uPA/tPA/plasmin/MMP proteolytic activities and thus maintains the tissue homeostasis. During wound healing, elevated levels of PAI-1 inhibit uPA/tPA/plasmin and plasmin-dependent MMP activities, and, thus, help expedite wound healing. In contrast to this scenario, under pathologic conditions, excessive PAI-1 contributes to excessive accumulation of collagen and other ECM protein in the wound area, and thus preserves scarring. While the level of PAI-1 is significantly elevated in fibrotic tissues, lack of PAI-1 protects different organs from fibrosis in response to injury-related profibrotic signals. Thus, PAI-1 is implicated in the pathology of fibrosis in different organs including the heart, lung, kidney, liver, and skin. Paradoxically, PAI-1 deficiency promotes spontaneous cardiac-selective fibrosis. In this review, we discuss the significance of PAI-1 in the pathogenesis of fibrosis in multiple organs.     

Crosstalk of hypoxia-mediated signaling pathways in upregulating plasminogen activator inhibitor-1 expression in keloid fibroblasts

Keloids are skin fibrotic conditions characterized by an excess accumulation of extracellular matrix (ECM) components secondary to trauma or surgical injuries. Previous studies have shown that plasminogen activator inhibitor-1 (PAI-1) can be upregulated by hypoxia and may contribute to keloid pathogenesis. In this study we investigate the signaling mechanisms involved in hypoxia-mediated PAI-1 expression in keloid fibroblasts. Using Northern and Western blot analysis, transient transfections, and pharmacological agents, we demonstrate that hypoxia-induced upregulation of PAI-1 expression is mainly controlled by hypoxia inducible factors-1alpha (HIF-1alpha) and that hypoxia leads to a rapid and transient activation of phosphatidylinositol-3-kinase/Akt (PI3-K/Akt) and extracellular signal-regulated kinases 1/2 (ERK1/2). Treatment of cells with PI-3K/Akt inhibitor (LY294002) and tyrosine protein kinase inhibitor (genistein) significantly attenuated hypoxia-induced PAI-1 mRNA and protein expression as well as promoter activation, apparently via an inhibition of the hypoxia-induced stabilization of HIF-1alpha protein, attenuation of the steady-state level of HIF-1alpha mRNA, and its DNA-binding activity. Even though disruption of ERK1/2 signaling pathway by PD98059 abolished hypoxia-induced PAI-1 promoter activation and mRNA/protein expression in keloid fibroblasts, it did not inhibit the hypoxia-mediated stabilization of HIF-1alpha protein and the steady-state level of HIF-1alpha mRNA nor its DNA binding activity. Our findings suggest that a combination of several signaling pathways, including ERK1/2, PI3-K/Akt, and protein tyrosine kinases (PTKs), may contribute to the hypoxia-mediated induction of PAI-1 expression via activation of HIF-1alpha in keloid fibroblasts.     

Role of endothelin-1 in the skin fibrosis of systemic sclerosis

Endothelin-1 (ET-1) acts as a key regulator of vasoconstriction and fibrosis. Many previous studies have focused on the role of ET-1 in scleroderma (systemic sclerosis, SSc).We investigated the effects of ET-1 on the production of extracellular matrix in SSc and normal skin fibroblasts. Primary cultured dermal fibroblasts from SSc patients and healthy controls were treated with ET-1 (25 ng/mL) for 0 min, 15 min, 1 h, 24 h, 48 h and 72 h, respectively. Our results showed that, in SSc fibroblasts, ET-1 upregulated collagen type I, connective tissue growth factor (CTGF), type I plasminogen activator inhibitor (PAI-1) and pAkt in a time-dependent manner within 72 h; in normal fibroblasts, 25 ng/mL ET-1 stimulation correlated with high levels of CTGF, PAI-1 and pAkt. The secretion of fibronectin (FN), collagen type I, and PAI-1 is markedly increased in the supernatant of both SSc fibroblasts and normal fibroblasts. Furthermore, ET-1 phosphorylates Smad2 and Smad3 in normal fibroblasts, but not in SSc fibroblasts. In conclusion, our results demonstrated that ET-1 may induce fibrosis in dermal fibroblasts through Akt signals.     

Ascochlorin suppresses TGF-β1-induced PAI-1 expression through the inhibition of phospho-EGFR in rat kidney fibroblast cells

Fibrosis is induced by the excessive and abnormal deposition of extracellular matrix (ECM) with various growth factors in tissues. Transforming growth factor-β1 (TGF-β1), the growth factor involved in fibrosis, modulates ECM synthesis and accumulation. TGF-β1 enhances the production of stimulators of ECM synthesis such as plasminogen activator inhibitor type 1 (PAI-1). As such, PAI-1 expression directly influences the proteolysis, invasion, and accumulation of ECM. It was shown in this study that ascochlorin, a prenylpenl antiobiotic, prevents the expression of profibrotic factors, such as PAI-1 and collagen type I, and that the TGF-β1-induced PAI-1 promoter activity is inhibited by ascochlorin. Ascochlorin abolishes the phosphorylation of the EGFR-MEK-ERK signaling pathway to regulate the TGF-β1-induced expression of PAI-1 without the inhibition of TβRII phosphorylation. Furthermore, the MEK inhibitor and EGFR siRNA block PAI-1 expression, and the Raf-1, MEK, and ERK signaling pathways for the regulation of PAI-1 expression. Ascochlorin suppresses the matrix metalloproteinases (MMPs) activity to activate the heparin-binding EGF-like growth factor (HB-EGF), to induce the phosphorylation of EGFR, and the MMPs inhibitor suppresses EGFR phosphorylation and the PAI-1 mRNA levels. These results suggest that ascochlorin prevents the expression of PAI-1 via the inhibition of an EGFR-dependent signal transduction pathway activated by MMPs.     

Asiatic Acid Isolated From Centella Asiatica Inhibits TGF-β1-induced Collagen Expression in Human Keloid Fibroblasts via PPAR-γ Activation

Keloids are fibroproliferative disorders characterized by exuberant extracellular matrix deposition and transforming growth factor (TGF)-β/Smad pathway plays a pivotal role in keloid pathogenesis. Centella asiatica extract has been applied in scar management for ages. As one of its major components, asiatic acid (AA) has been recently reported to inhibit liver fibrosis by blocking TGF-β/Smad pathway. However, its effect on keloid remains unknown. In order to investigate the effects of AA on cell proliferation, invasion and collagen synthesis, normal and keloid fibroblasts were exposed to TGF-β1 with or without AA. Relevant experiments including 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, 5-ethynyl-2-deoxyuridine (EdU) incorporation assay, Transwell invasion assay, enzyme-linked immunosorbent assay, Western blot, quantitative polymerase chain reaction and RNA interference assay were conducted. As a result, keloid fibroblasts showed higher responsiveness to TGF-β1 stimulation than normal fibroblasts in terms of invasion and collagen synthesis. AA could suppress TGF-β1-induced expression of collagen type I, inhibit Smad 2/3 phosphorylation and plasminogen activator inhibitor-1 (PAI-1) expression, while elevate Smad 7 protein level. Noteworthy, the effects of AA on keloid fibroblasts could be abrogated by PPAR-γ antagonist GW9662 and by silencing of PPAR-γ. The present study demonstrated that AA inhibited TGF-β1-induced collagen and PAI-1 expression in keloid fibroblasts through PPAR-γ activation, which suggested that AA was one of the active constituents of C. asiatica responsible for keloid management, and could be included in the arsenal for combating against keloid.     

Expression of urokinase plasminogen activator, its receptor and plasminogen activator inhibitor type 1 in different types of atherosclerotic lesion in human aorta

The role of plasminogen activators in the regulation of key processes of atherosclerosis progression stays unclear. The aim of this study was to evaluate the expression of urokinase plasminogen activator (uPA), its receptor (uPAR) and the plasminogen activator inhibitor type 1 (PAI-1) in human aorta, and to balance them with the stage of atherosclerotic lesion. We have shown that uPA and uPAR in normal aorta are mostly expressed by intimal smooth muscle cells. The expression of these proteins was up-regulated in diseased aorta compared to normal artery. The most part of cells in both fatty streak and fibro-fatty lesion were monocytes/macrophages, and about 60% of these cells expressed uPA and its receptor. PAI-1 was mostly localized on the lumonal part of the aorta and in the extracellular matrix of the intima. We observed a moderate increase of PAI-1 expression in atherosclerotic lesion. Thus, our data indicate participation of plasminogen system in atherogenesis.     

Collagen modulates gene activation of plasminogen activator system molecules

Skin extracellular matrix (ECM) molecules regulate a variety of cellular activities, including cell movement, which are central to wound healing and metastasis. Regulated cell movement is modulated by proteases and their associated molecules, including the serine proteases urinary-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) and their inhibitors (PAIs). As a result of wounding and loss of basement membrane structure, epidermal keratinocytes can become exposed to collagen. To test the hypothesis that during wounding, exposed collagen, the most abundant ECM molecule in the skin, regulates keratinocyte PA and PAI gene expression, we utilized an in vitro model in which activated keratinocytes were cultured in dishes coated with collagen or other ECM substrates. tPA, uPA, and PAI-1 mRNA and enzymatic activity were detected when activated keratinocytes attached to fibronectin, vitronectin, collagen IV, and RGD peptide. In contrast, adhesion to collagen I and collagen III completely suppressed expression of PAI-1 mRNA and protein and further increased tPA expression and activity. Similarly, keratinocyte adhesion to laminin-1 suppressed PAI-1 mRNA and protein expression and increased tPA activity. The suppressive effect of collagen I on PAI-1 gene induction was dependent on the maintenance of its native fibrillar structure. Thus, it would appear that collagen- and laminin-regulated gene expression of molecules associated with plasminogen activation provides an additional dimension in the regulation of cell movement and matrix remodeling in skin wound healing.     

Thrombin neutralizes plasminogen activator inhibitor 1 (PAI-1) that is complexed with vitronectin in the endothelial cell matrix

Vitronectin endows plasminogen activator inhibitor 1 (PAI-1), the fast-acting inhibitor of both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), with additional thrombin inhibitory properties. In view of the apparent association between PAI-1 and vitronectin in the endothelial cell matrix (ECM), we analyzed the interaction between PAI-1 and thrombin in this environment. Upon incubating 125I-labeled alpha-thrombin with endothelial cell matrix (ECM), the protease formed SDS-stable complexes exclusively with PAI-1, with subsequent release of these complexes into the supernatant. Vitronectin was required as a cofactor for the association between PAI-1 and thrombin in ECM. Metabolic labeling of endothelial cell proteins, followed by incubation of ECM with t-PA, u-PA, or thrombin, indicated that all three proteases depleted PAI-1 from ECM by complex formation and proteolytic cleavage. Proteolytically inactive thrombin as well as anticoagulant thrombin, i.e., thrombin in complex with its endothelial cell surface receptor thrombomodulin, did not neutralize PAI-1, emphasizing that the procoagulant moiety of thrombin is required for a functional interaction with PAI-1. A physiological implication of our findings may be related to the mutual neutralization of both PAI-1 and thrombin, providing a new link between plasminogen activation and the coagulation system. Evidence is provided that in ECM, procoagulant thrombin may promote plasminogen activator activity by inactivating PAI-1.     

The role of urokinase in vascular cell migration and in regulation of growth and branching of capillaries

The urokinase system, represented by a plasminogen activator of urokinase type (urokinase, uPA), urokinase receptor (uPAR), and inhibitors of plasminogen activator (PAI-1 and PAI-2), plays an important role in the regulation of vascular wall functioning. Urokinase signaling initiates proteolytic cascade and degradation of the extracellular matrix; and also activates intracellular signaling in vascular cells. This study is the first to reveal a urokinase-mediated fundamental mechanism that regulates the growth trajectory and branching morphogenesis of blood vessels. This mechanism may be of particular importance during vessel growth in early embryogenesis and in the adult during tissue regeneration.     

Matrix-bound plasminogen activator inhibitor type 1 inhibits the invasion of human monocytes into interstitial tissue.

Invasion of tissue by monocytes in the course of cellular immune reactions is a multistep process that is thought to be based on the action of urokinase type plasminogen activator (u-PA), an ubiquitous serine protease able to convert the zymogen plasminogen into the active protease plasmin. Expression and occupation of urokinase-type plasminogen activator receptors (u-PA-R) are known to be up-regulated by IFN-gamma and TNF-alpha, and endogenously occupied u-PA-R were found to be instrumental in monocyte invasiveness. We used the amnion invasion assay to investigate whether monocyte invasiveness is affected by matrix-bound plasminogen activator inhibitors (PAI) and by fluid phase u-PA. We show in this study that preincubation of amnion membranes with 1.5 U/cm2 PAI-1 decreases invasion of IFN-gamma activated monocytes by 70% compared with controls. Anti-vitronectin antibodies, which block PAI-1 binding to the matrix, abrogate the inhibitory effect of PAI-1 on monocyte invasiveness, indicating that active PAI-1 is bound via matrix-associated vitronectin. In contrast, preincubation of the amnion membrane with PAI-2 which does not bind to the extracellular matrix has no effect on monocyte invasiveness. Finally, the inhibitory action of matrix-bound PAI-1 can be abrogated by addition of 5 IU/ml u-PA to the monocytes in the invasion chamber. These findings indicate that monocyte invasiveness might be regulated not only by expression and occupation of u-PA-R but also by matrix-bound PAI-1.     

LRP1 regulates remodeling of the extracellular matrix by fibroblasts

Low density lipoprotein receptor-related protein (LRP1) is an endocytic receptor for diverse proteases, protease inhibitors, and other plasma membrane proteins, including the urokinase receptor (uPAR). LRP1 also functions in cell-signaling and regulates gene expression. The goal of this study was to determine whether LRP1 regulates remodeling of provisional extracellular matrix (ECM) by fibroblasts. To address this problem, we utilized an in vitro model in which type I collagen was reconstituted and overlaid with fibronectin. Either the collagen or fibronectin was fluorescently-labeled. ECM remodeling by fibroblasts deficient in LRP1, uPAR, or MT1-MMP was studied. MT1-MMP was required for efficient remodeling of the deep collagen layer but not involved in fibronectin remodeling. Instead, fibronectin was remodeled by a system that required urokinase-type plasminogen activator (uPA), uPAR, and exogenously-added plasminogen. LRP1 markedly inhibited fibronectin remodeling by regulating cell-surface uPAR and plasminogen activation. LRP1 also regulated remodeling of the deep collagen layer but not by controlling MT1-MMP. Instead, LRP1 deficiency or inhibition de-repressed a secondary pathway for collagen remodeling, which was active in MT1-MMP-deficient cells but not in uPAR-deficient cells. These results demonstrate that LRP1 regulates ECM remodeling principally by repressing pathways that require plasminogen activation by uPA in association with uPAR.     

Vitronectin regulates the synthesis and localization of urokinase-type plasminogen activator in HT-1080 cells.

The effect of extracellular matrix composition on the location, amount, and activity of cell-associated urokinase-type plasminogen activator was tested using HT-1080 cells adherent to either fibronectin or vitronectin. Specific immunoprecipitation of newly synthesized urokinase indicated that cells adherent to fibronectin synthesized 2-3-fold more urokinase than cells adherent to vitronectin. Complexes of urokinase and plasminogen activator inhibitor type 1 (PAI-1) were detected in cell layers of vitronectin-adherent but not fibronectin-adherent cells. Inhibition of PAI-1 using a neutralizing monoclonal antibody resulted in a 3-fold increase in urokinase enzymatic activity on vitronectin adherent cells. Urokinase activity on fibronectin adherent cells was only slightly increased following PAI-1 neutralization. Examination of both HT-1080 and normal human fibroblast cells by immunofluorescent microscopy localized urokinase-type plasminogen activator to discrete, focal areas underneath cells adherent to vitronectin. Urokinase was not detectable by immunofluorescence on cells adherent to fibronectin. The addition of exogenous prourokinase to locate urokinase receptors on adherent HT-1080 cells indicated that the focal localization of cell-surface urokinase resulted from the clustering of urokinase receptors following adhesion to vitronectin but not fibronectin-coated substrates. These results suggest that vitronectin can contribute to the control of cell-surface plasmin activity by regulating the synthesis of urokinase and directing the localization of urokinase receptors.     

PAI-1 deficiency reduces liver fibrosis after bile duct ligation in mice through activation of tPA

Plasminogen activator inhibitor-1 (PAI-1) increases injury in several liver, lung and kidney disease models. The objective of this investigation was to assess the effect of PAI-1 deficiency on cholestatic liver fibrosis and determine PAI-1 influenced fibrogenic mechanisms. We found that PAI-1(-/-) mice had less fibrosis than wild type (WT) mice after bile duct ligation. This change correlated with increased tissue-type plasminogen activator (tPA) activity, and increased matrix metalloproteinase-9 (MMP-9), but not MMP-2 activity. Furthermore, there was increased activation of the tPA substrate hepatocyte growth factor (HGF), a known anti-fibrogenic protein. In contrast, there was no difference in hepatic urokinase plasminogen activator (uPA) or plasmin activities between PAI-1(-/-) and WT mice. There was also no difference in the level of transforming growth factor beta 1 (TGF-beta1), stellate cell activation or collagen production between WT and PAI-1(-/-) animals. In conclusion, PAI-1 deficiency reduces hepatic fibrosis after bile duct obstruction mainly through the activation of tPA and HGF.     

The prognostic value of vascular endothelial growth factor, urokinase plasminogen activator and plasminogen activator inhibitor-1 in node-negative breast cancer

The vascular endothelial growth factor (VEGF) and the plasminogen activator system play an essential role in solid tumor angiogenesis and in tumor invasion and metastasis. In the present study we investigated the relationship between patient outcome and levels of VEGF, urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) in tumor cytosols of 196 node-negative primary invasive breast cancer patients who did not receive any adjuvant therapy. The median follow-up was 65 months. VEGF, uPA and PAI-1 were measured by commercially available enzyme-linked immunosorbent assays. Cox's univariate analysis showed that pT (p = 0.0007), uPA (p = 0.0156) and PAI-1 (p = 0.0015) had a significant impact on relapse-free survival, whereas VEGF did not have any prognostic value (p = 0.18). Bivariate analysis showed significant interactions between uPA and PAI-1 (p = 0.0035) and between VEGF and PAI-1 (p = 0.006). Our study confirms that uPA and PAI-1 cytosol levels can be considered as prognostic factors for relapse-free survival in node-negative breast cancer. Moreover, the interaction between VEGF and PAI-1 warrants further investigation into the relationship between the biomarkers of angiogenesis and those of the protease cascade.     

Hypoxia-induced HIF-1 alpha accumulation is augmented in a co-culture of keloid fibroblasts and human mast cells: involvement of ERK1/2 and PI-3K/Akt

Keloids represent a prolonged inflammatory fibrotic state with areas that display distinctive histological features characterized by an abundant extracellular matrix stroma, a local infiltration of inflammatory cells including mast cells, and a milieu of enriched cytokines. Previous studies from our laboratory demonstrated an intrinsic higher level of HIF-1alpha and VEGF protein expression in keloid tissues compared with their adjacent unremarkable skins. To further investigate the mechanisms underlying the elevated expression of HIF-1alpha and VEGF in keloids, we exposed a co-culture of keloid fibroblasts and mast cells (HMC-1) to hypoxic conditions and studied the expression of HIF-1alpha and its target gene, VEGF. Our results showed that hypoxia-dependent HIF-1alpha protein accumulation and VEGF expression is augmented in keloid fibroblasts when co-cultured with HMC-1 cells under the condition where direct cell-cell contact is allowed. But such augmentation is not observed in the transwell co-culture system whereas fibroblasts and HMC-1 cells were separated by a porous membrane. Our results also indicated that the enhancement of hypoxia-mediated activation of ERK1/2 and Akt requires direct cell-cell interaction between mast cells and keloid fibroblasts, and activation of both ERK1/2 and Akt is involved in the hypoxia-dependent HIF-1alpha protein accumulation and VEGF expression in the co-culture system. These findings suggest that under hypoxic conditions mast cells may contribute, at least in part, to an elevated expression of HIF-1alpha and VEGF protein in keloids via direct cell-cell interaction with fibroblasts.