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.     

Role of plasminogen activators in peritoneal adhesion formation

Intra-abdominal adhesion formation is a major complication of serosal repair following surgery, ischaemia or infection, leading to conditions such as intestinal obstruction and infertility. It has been proposed that the persistence of fibrin, due to impaired plasminogen activator activity, results in the formation of adhesions between damaged serosal surfaces. This study aimed to assess the role of fibrinolysis in adhesion formation using mice deficient in either of the plasminogen activator proteases, tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA). We hypothesize that, following serosal injury, mice with decreased peritoneal fibrinolytic activity will be more susceptible to adhesion formation. Adhesion formation was induced in tPA- and uPA-deficient and wild-type mice following either surgical trauma to the serosa with haemorrhage and acute or chronic intraperitoneal inflammation. Adhesion formation was assessed from 1 to 4 weeks post-injury. Mice deficient in tPA were more susceptible to adhesion formation following both a surgical insult and a chronic inflammatory episode compared with uPA-deficient and wild-type mice. In addition, the time of maximal adhesion formation varied depending on the nature of the initial insult. It is proposed that the persistence of fibrin due to decreased tPA activity following surgery or chronic inflammation plays a major role in peritoneal adhesion formation.     

Endogenous tissue-type plasminogen activator is protective during Escherichia coli-induced abdominal sepsis in mice

Sepsis is associated with enhanced production of tissue-type plasminogen activator (tPA). We investigated the function of endogenous tPA in the immune responses to Escherichia coli-induced abdominal sepsis using tPA gene-deficient (tPA(-/-)) and normal wild-type (WT) mice. tPA(-/-) mice demonstrated an impaired defense against E. coli peritonitis as indicated by higher bacterial loads at the primary site of the infection, enhanced dissemination, and reduced survival. The protective function of tPA was independent of plasmin since plasminogen gene-deficient (Plg(-/-)) mice were indistinguishable from WT mice. Relative to WT mice, tPA(-/-) mice demonstrated similar neutrophil counts in the peritoneal cavity despite much higher bacterial loads and higher local concentrations of neutrophil attracting chemokines, suggesting a reduced migratory response. In line, tPA(-/-) mice demonstrated a reduced thioglycolate-induced neutrophil influx into the peritoneal cavity and i.p. injection of WT mice with a replication-defective adenoviral vector expressing tPA caused an enhanced cell migration to the peritoneal cavity during E. coli peritonitis. These findings identify a novel protective function of tPA in abdominal sepsis caused by E. coli that seems independent of its role in the generation of plasmin.     

Tissue-type plasminogen activator is not necessary for platelet-derived growth factor-c activation

Platelet-derived growth factors (PDGFs) are critical for development; their over-expression is associated with fibrogenesis. Full-length PDGF-C is secreted as an inactive dimer, requiring cleavage to allow receptor binding. Previous studies indicate that tissue-type plasminogen activator (tPA) is the specific protease that performs this cleavage; in vivo confirmation is lacking. We demonstrate that primary hepatocytes from tpa KO mice produce less cleaved active PDGF-CC than do wild type hepatocytes, suggesting that tPA is critical for in vitro activation of this growth factor. We developed mice that over-express full-length human PDGF-C in the liver; these mice develop progressive liver fibrosis. To test whether tPA is important for cleavage and activation of PDGF-C in vivo, we intercrossed PDGF-C transgenic (Tg) and tpa knock-out (KO) mice, anticipating that lack of tPA would result in decreased fibrosis due to lack of hPDGF-C cleavage. To measure levels of cleaved, dimerized PDGF-CC in sera, we developed an ELISA that specifically detects cleaved PDGF-CC. We report that the absence of tpa does not affect the phenotype of `PDGF-C Tg mice. PDGF-C Tg mice lacking tPA have high serum levels of cleaved growth factor, significant liver fibrosis, and gene expression alterations similar to those of PDGF-C Tg mice with intact tPA. Furthermore, urokinase plasminogen activator and plasminogen activator inhibitor-1 expression are increased in PDGF-C Tg; tpa KO mice. Our ELISA data suggest a difference between in vitro and in vivo activation of this growth factor, and our mouse model confirms that multiple proteases cleave and activate PDGF-C in vivo.     

Tissue plasminogen activator-mediated fibrinolysis protects against axonal degeneration and demyelination after sciatic nerve injury

Tissue plasminogen activator (tPA) is a serine protease that converts plasminogen to plasmin and can trigger the degradation of extracellular matrix proteins. In the nervous system, under noninflammatory conditions, tPA contributes to excitotoxic neuronal death, probably through degradation of laminin. To evaluate the contribution of extracellular proteolysis in inflammatory neuronal degeneration, we performed sciatic nerve injury in mice. Proteolytic activity was increased in the nerve after injury, and this activity was primarily because of Schwann cell-produced tPA. To identify whether tPA release after nerve damage played a beneficial or deleterious role, we crushed the sciatic nerve of mice deficient for tPA. Axonal demyelination was exacerbated in the absence of tPA or plasminogen, indicating that tPA has a protective role in nerve injury, and that this protective effect is due to its proteolytic action on plasminogen. Axonal damage was correlated with increased fibrin(ogen) deposition, suggesting that this protein might play a role in neuronal injury. Consistent with this idea, the increased axonal degeneration phenotype in tPA- or plasminogen-deficient mice was ameliorated by genetic or pharmacological depletion of fibrinogen, identifying fibrin as the plasmin substrate in the nervous system under inflammatory axonal damage. This study shows that fibrin deposition exacerbates axonal injury, and that induction of an extracellular proteolytic cascade is a beneficial response of the tissue to remove fibrin. tPA/plasmin-mediated fibrinolysis may be a widespread protective mechanism in neuroinflammatory pathologies.     

Proteolytic fragments of laminin promote excitotoxic neurodegeneration by up-regulation of the KA1 subunit of the kainate receptor

Degradation of the extracellular matrix (ECM) protein laminin contributes to excitotoxic cell death in the hippocampus, but the mechanism of this effect is unknown. To study this process, we disrupted laminin γ1 (lamγ1) expression in the hippocampus. Lamγ1 knockout (KO) and control mice had similar basal expression of kainate (KA) receptors, but the lamγ1 KO mice were resistant to KA-induced neuronal death. After KA injection, KA1 subunit levels increased in control mice but were unchanged in lamγ1 KO mice. KA1 levels in tissue plasminogen activator (tPA)–KO mice were also unchanged after KA, indicating that both tPA and laminin were necessary for KA1 up-regulation after KA injection. Infusion of plasmin-digested laminin-1 into the hippocampus of lamγ1 or tPA KO mice restored KA1 up-regulation and KA-induced neuronal degeneration. Interfering with KA1 function with a specific anti-KA1 antibody protected against KA-induced neuronal death both in vitro and in vivo. These results demonstrate a novel pathway for neurodegeneration involving proteolysis of the ECM and KA1 KA receptor subunit up-regulation.     

Disruption of tissue plasminogen activator gene reduces macrophage migration

Tissue plasminogen activator (tPA) is an essential component of the proteolytic cascade that lyses blood clots. Various studies also suggest that tPA plays important roles in peripheral nerve regeneration. Here we show that disruption of tPA gene reduces macrophage migration after sciatic nerve injury in mice. Moreover, lack of tPA activity attenuates migrating ability of macrophages and affects MMP-9 expression and activity in macrophages in vitro. Addition of ethylenediaminetetraacetic acid (EDTA), which inhibits MMPs, abolished the differences of migration ability of macrophages between tPA(+/+) and tPA(-/-) mice. Axonal regeneration is correlated with the increase of macrophage migration, suggesting that tPA may help create a beneficial environment for axonal regeneration through promoting macrophage infiltration. This study shows that tPA may play a role in nerve regeneration through regulating the migration ability of macrophages. This function of tPA may depend on, at least in part, upregulating MMP-9 expression and activity in macrophages.     

tPA Regulates Neurite Outgrowth by Phosphorylation of LRP5/6 in Neural Progenitor Cells

Despite the important role of tissue plasminogen activator (tPA) as a neuromodulator in neurons, microglia, and astrocytes, its role in neural progenitor cell (NPC) development is not clear yet. We identified that tPA is highly expressed in NPCs compared with neurons. Inhibition of tPA activity or expression using tPA stop, PAI-1, or tPA siRNA inhibited neurite outgrowth from NPCs, while overexpression or addition of exogenous tPA increased neurite outgrowth. The expression of Wnt and β-catenin as well as phosphorylation of LRP5 and LRP6, which has been implicated in Wnt–β-catenin signaling, was rapidly increased after tPA treatment and was decreased by tPA siRNA transfection. Knockdown of β-catenin or LRP5/6 expression by siRNA prevented tPA-induced neurite extension. NPCs obtained from tPA KO mice showed impaired neurite outgrowth compared with WT NPCs. In ischemic rat brains, axon density was higher in the brains transplanted with WT NPCs than in those with tPA KO NPCs, suggesting increased axonal sprouting by NPC-derived tPA. tPA-mediated regulation of neuronal maturation in NPCs may play an important role during development and in regenerative conditions.     

Components of the Plasminogen Activation System Promote Engraftment of Porous Polyethylene Biomaterial via Common and Distinct Effects

Rapid fibrovascularization is a prerequisite for successful biomaterial engraftment. In addition to their well-known roles in fibrinolysis, urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA) or their inhibitor plasminogen activator inhibitor-1 (PAI-1) have recently been implicated as individual mediators in non-fibrinolytic processes, including cell adhesion, migration, and proliferation. Since these events are critical for fibrovascularization of biomaterial, we hypothesized that the components of the plasminogen activation system contribute to biomaterial engraftment. Employing in vivo and ex vivo microscopy techniques, vessel and collagen network formation within porous polyethylene (PPE) implants engrafted into dorsal skinfold chambers were found to be significantly impaired in uPA-, tPA-, or PAI-1-deficient mice. Consequently, the force required for mechanical disintegration of the implants out of the host tissue was significantly lower in the mutant mice than in wild-type controls. Conversely, surface coating with recombinant uPA, tPA, non-catalytic uPA, or PAI-1, but not with non-catalytic tPA, accelerated implant vascularization in wild-type mice. Thus, uPA, tPA, and PAI-1 contribute to the fibrovascularization of PPE implants through common and distinct effects. As clinical perspective, surface coating with recombinant uPA, tPA, or PAI-1 might provide a novel strategy for accelerating the vascularization of this biomaterial.     

CXCR2/CXCR2 ligand biology during lung transplant ischemia-reperfusion injury

Lung transplantation is a therapeutic option for a number of end-stage pulmonary disorders. Early lung allograft dysfunction (ischemia-reperfusion injury) continues to be the most common cause of early mortality after lung transplantation and a significant risk factor for the development of bronchiolitis obliterans syndrome. Ischemia-reperfusion injury is characterized histopathologically by lung edema and a neutrophil predominate leukocyte extravasation. The specific mechanism(s) that recruit leukocytes to the lung during post-lung transplantation ischemia-reperfusion injury have not been fully elucidated. Because the ELR+ CXC chemokines are potent neutrophil chemoattractants, we investigated their role during post-lung transplantation ischemic-reperfusion injury. We found elevated levels of multiple ELR+ CXC chemokines in human bronchoalveolar lavage fluid from patients with ischemia-reperfusion injury. Proof of concept studies using a rat orthotopic lung transplantation model of "cold" ischemic-reperfusion injury demonstrated an increase in lung graft neutrophil sequestration and injury. In addition, lung expression of CXCL1, CXCL2/3, and their shared receptor CXCR2 paralleled lung neutrophil infiltration and injury. Importantly, inhibition of CXCR2/CXCR2 ligand interactions in vivo led to a marked reduction in lung neutrophil sequestration and graft injury. Taken together these experiments support the notion that increased expression of ELR+ CXC chemokines and their interaction with CXCR2 plays an important role in the pathogenesis of post-lung transplantation cold ischemia-reperfusion injury.     

Tissue plasminogen activator is a potent activator of PDGF-CC

Tissue plasminogen activator (tPA) is a serine protease involved in the degradation of blood clots through the activation of plasminogen to plasmin. Here we report on the identification of tPA as a specific protease able to activate platelet-derived growth factor C (PDGF-C). The newly identified PDGF-C is secreted as a latent dimeric factor (PDGF-CC) that upon proteolytic removal of the N-terminal CUB domains becomes a PDGF receptor alpha agonist. The CUB domains in PDGF-CC directly interact with tPA, and fibroblasts from tPA-deficient mice fail to activate latent PDGF-CC. We further demonstrate that growth of primary fibroblasts in culture is dependent on a tPA-mediated cleavage of latent PDGF-CC, generating a growth stimulatory loop. Immunohistochemical analysis showed similar expression patterns of PDGF-C and tPA in developing mouse embryos and in tumors, indicating both autocrine and paracrine modes of activation of PDGF receptor-mediated signaling pathways. The identification of tPA as an activator of PDGF signaling establishes a novel role for the protease in normal and pathological tissue growth and maintenance, distinct from its well-known role in plasminogen activation and fibrinolysis.     

p75 neurotrophin receptor regulates tissue fibrosis through inhibition of plasminogen activation via a PDE4/cAMP/PKA pathway

Clearance of fibrin through proteolytic degradation is a critical step of matrix remodeling that contributes to tissue repair in a variety of pathological conditions, such as stroke, atherosclerosis, and pulmonary disease. However, the molecular mechanisms that regulate fibrin deposition are not known. Here, we report that the p75 neurotrophin receptor (p75^NTR), a TNF receptor superfamily member up-regulated after tissue injury, blocks fibrinolysis by down-regulating the serine protease, tissue plasminogen activator (tPA), and up-regulating plasminogen activator inhibitor-1 (PAI-1). We have discovered a new mechanism in which phosphodiesterase PDE4A4/5 interacts with p75^NTR to enhance cAMP degradation. The p75^NTR-dependent down-regulation of cAMP results in a decrease in extracellular proteolytic activity. This mechanism is supported in vivo in p75^NTR-deficient mice, which show increased proteolysis after sciatic nerve injury and lung fibrosis. Our results reveal a novel pathogenic mechanism by which p75^NTR regulates degradation of cAMP and perpetuates scar formation after injury.     

Gastrointestinal ischemia-reperfusion injury is lectin complement pathway dependent without involving C1q

Complement activation plays an important role in local and remote tissue injury associated with gastrointestinal ischemia-reperfusion (GI/R). The role of the classical and lectin complement pathways in GI/R injury was evaluated using C1q-deficient (C1q KO), MBL-A/C-deficient (MBL-null), complement factor 2- and factor B-deficient (C2/fB KO), and wild-type (WT) mice. Gastrointestinal ischemia (20 min), followed by 3-h reperfusion, induced intestinal and lung injury in C1q KO and WT mice, but not in C2/fB KO mice. Addition of human C2 to C2/fB KO mice significantly restored GI/R injury, demonstrating that GI/R injury is mediated via the lectin and/or classical pathway. Tissue C3 deposition in C1q KO and WT, but not C2/fB KO, mice after GI/R demonstrated that complement was activated in C1q KO mice. GI/R significantly increased serum alanine aminotransferase, gastrointestinal barrier dysfunction, and neutrophil infiltration into the lung and gut in C1q KO and WT, but not C2/fB KO, mice. MBL-null mice displayed little gut injury after GI/R, but lung injury was present. Addition of recombinant human MBL (rhuMBL) to MBL-null mice significantly increased injury compared with MBL-null mice after GI/R and was reversed by anti-MBL mAb treatment. However, MBL-null mice were not protected from secondary lung injury after GI/R. These data demonstrate that C2 and MBL, but not C1q, are necessary for gut injury after GI/R. Lung injury in mice after GI/R is MBL and C1q independent, but C2 dependent, suggesting a potential role for ficolins in this model.     

Characterizing the role of tissue-type plasminogen activator in a mouse model of Group A streptococcal infection

Plasmin(ogen) acquisition is critical for invasive disease initiation by Streptococcus pyogenes (GAS). Host urokinase plasminogen activator (uPA) plays a role in mediating plasminogen activation for GAS dissemination, however the contribution of tissue-type plasminogen activator (tPA) to GAS virulence is unknown. Using novel tPA-deficient ALBPLG1 mice, our study revealed no difference in mouse survival, bacterial dissemination or the pathology of GAS infection in the absence of tPA in AlbPLG1/tPA^?/? mice compared to AlbPLG1 mice. This study suggests that tPA has a limited role in this humanized model of GAS infection, further highlighting the importance of its counterpart uPA in GAS disease.     

Tissue-plasminogen activator stimulates endothelial cell migration in wound assays

The ability of tissue plasminogen activator (tPA) to induce human umbilical vein endothelial (HUVE) cell migration was studied using an in vitro, serum-free wound assay system. At pharmacological doses, tPA stimulated HUVE cell migration dose-dependently. Treatment of cells with epsilon amino caproic acid (EACA) to detach cell-surface and extracellular matrix bound plasminogen, which could lead to plasmin generation, resulted in increased HUVEcell migration on stimulation with tPA.Plasminogen activator inhibitor-1 (PAI-1), a natural plasminogen activator inhibitor, abolished tPA-induced HUVEcell migration. These results demonstrate for the first time that tPA is capable of stimulating endothelial cell migration in wound assays and this effect is susceptible to PAI-1 inhibition.     

Tissue plasminogen activator and its receptor in the human amnion, chorion, and decidua at preterm and term

The plasminogen activator system consists of two proteins: tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), which act upon their specific receptors to generate plasmin from plasminogen located on the cell surface. Plasmin then acts directly and indirectly to degrade the components of the extracellular matrix (ECM). This process is likely to be important in the normal turnover of the ECM of fetal membranes and in its premature weakening in preterm premature rupture of the fetal membranes. Quantitative Northern analysis and in situ hybridization have shown that the decidua expresses mRNA for tPA. However, the immunolocalized tPA protein was most strongly associated with the amnion and chorion, as was its receptor annexin II, suggesting that the amnion and chorion are the targets for decidual tPA. At term, decidual tPA expression was unaffected by labor, and the tPA receptor was elevated both before and after labor. At preterm, the converse was found: decidual tPA expression was significantly (p < 0. 05) up-regulated by labor, but the tPA receptor was not. The results suggest that the generation of plasmin at term would be controlled by an increased concentration of the tPA receptor in the amnion and chorion, whereas at preterm a pathological increase in plasmin would be generated by an overexpression of tPA, initiated by labor.     

Regulation of lipopolysaccharide-induced lung inflammation by plasminogen activator Inhibitor-1 through a JNK-mediated pathway

The neutrophil is of undoubted importance in lung inflammation after exposure to LPS. We have shown recently that systemic inhibition of JNK decreased neutrophil recruitment to the lung after exposure to LPS, although the mechanisms underlying this inhibition are incompletely understood. As plasminogen activator inhibitor-1 (PAI-1) accentuates cell migration, with JNK activation recently shown to up-regulate PAI-1 expression, this suggested that systemic JNK inhibition may down-regulate LPS-induced pulmonary neutrophil recruitment through a decrease in PAI-1 expression. We show in this study that exposure of mice to aerosolized LPS increased PAI-1 expression in the lung and alveolar compartment, which was decreased by pretreatment with the JNK inhibitor SP600125. Exogenous, intratracheally administered PAI-1 prevented the inhibition of pulmonary neutrophil recruitment in the setting of systemic JNK inhibition, thereby suggesting a role for PAI-1 in the JNK-mediated pathway regulating LPS-induced neutrophil recruitment. In addition, PAI-1(-/-) mice had a decrease in neutrophil recruitment to the alveolar compartment after exposure to LPS, compared with wild-type controls, further suggesting a role for PAI-1 in LPS-induced lung inflammation. An increase in the intravascular level of KC is a likely mechanism for the inhibition of pulmonary neutrophil recruitment after LPS exposure in the setting of decreased PAI-1 expression, as systemic KC levels after exposure to LPS were increased in PAI-1-deficient mice and in mice pretreated with SP600125, with augmentation of intravascular KC levels inhibiting neutrophil recruitment to the lung after exposure to LPS.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates the expression and the release of tissue plasminogen activator (tPA) in neuronal cells: involvement of tPA in the neuroprotective effect of PACAP

Pituitary adenylate cyclase-activating polypeptide (PACAP) and tissue plasminogen activator (tPA) play important roles in neuronal migration and survival. However, a direct link between the neurotrophic effects of PACAP and tPA has never been investigated. In this study, we show that, in PC12 cells, PACAP induced a 9.85-fold increase in tPA gene expression through activation of the protein kinase A- and protein kinase C-dependent signaling pathways. In immature cerebellar granule neurons (CGN), PACAP stimulated tPA mRNA expression and release of proteolytically active tPA. Immunocytochemical labeling revealed the presence of tPA in the cytoplasm and processes of cultured CGN. The inhibitory effect of PACAP on CGN motility was not affected by the tPA substrate plasminogen or the tPA inhibitor plasminogen activator inhibitor-1. In contrast, plasminogen activator inhibitor-1 significantly reduced the stimulatory effect of PACAP on CGN survival. Altogether, these data indicate that tPA gene expression is activated by PACAP in both tumoral and normal neuronal cells. The present study also demonstrates that PACAP stimulates the release of tPA which promotes CGN survival by a mechanism dependent of its proteolytic activity.     

Inducible lung-specific urokinase expression reduces fibrosis and mortality after lung injury in mice

Plasminogen activator inhibitor-1 (PAI-1)-deficient transgenic mice have improved survival and less fibrosis after intratracheal bleomycin instillation. We hypothesize that PAI-1 deficiency limits scarring through unopposed plasminogen activation. If this is indeed true, then we would expect increased urokinase-type plasminogen activator (uPA) expression to result in a similar reduction in scarring and improvement in mortality. To test our hypothesis, using the tetracycline gene regulatory system, we have generated a transgenic mouse model with the features of inducible, lung-specific uPA production. After doxycycline administration, these transgenic animals expressed increased levels of uPA in their bronchoalveolar lavage (BAL) fluid that accelerated intrapulmonary fibrin clearance. Importantly, this increased plasminogen activator production led to a reduction in both lung collagen accumulation and mortality after bleomycin-induced injury. These results suggest that PAI-1 deficiency does protect against the effects of bleomycin-induced lung injury through unopposed plasmin generation. By allowing the manipulation of plasminogen activation at different phases of the fibrotic process, this model will serve as a powerful tool in further investigations into the pathogenesis of pulmonary fibrosis.