Mechanisms of cardiac fibrosis induced by urokinase plasminogen activator

Human hearts with end-stage failure and fibrosis have macrophage accumulation and elevated plasminogen activator activity. However, the mechanisms that link macrophage accumulation and plasminogen activator activity with cardiac fibrosis are unclear. We previously reported that mice with macrophage-targeted overexpression of urokinase plasminogen activator (SR-uPA+/o mice) develop cardiac macrophage accumulation by 5 weeks of age and cardiac fibrosis by 15 weeks. We used SR-uPA+/o mice to investigate mechanisms through which macrophage-expressed uPA causes cardiac macrophage accumulation and fibrosis. We hypothesized that: 1) macrophage accumulation and cardiac fibrosis in SR-uPA+/o mice are dependent on localization of uPA by the uPA receptor (uPAR); 2) activation of plasminogen by uPA and subsequent activation of transforming growth factor-beta1 (TGF-beta1) and matrix metalloproteinase (MMP)-2 and -9 by plasmin are critical pathways through which uPA-expressing macrophages accumulate in the heart and cause fibrosis; and 3) uPA-induced cardiac fibrosis can be attenuated by treatment with verapamil. To test these hypotheses, we bred the SR-uPA+/o transgene into mice deficient in either uPAR or plasminogen and measured cardiac macrophage accumulation and fibrosis. We also measured cardiac TGF-beta1 protein (total and active), Smad2 phosphorylation, and MMP activity after the onset of macrophage accumulation but before the onset of cardiac fibrosis. Finally, we treated mice with verapamil. Our studies revealed that plasminogen is necessary for uPA-induced cardiac fibrosis and macrophage accumulation but uPAR is not. We did not detect plasmin-mediated activation of TGF-beta1, MMP-2, or MMP-9 in hearts of SR-uPA+/o mice. However, verapamil treatment significantly attenuated both cardiac fibrosis and macrophage accumulation.     

Murine renal allografts: spontaneous acceptance is associated with regulated T cell-mediated immunity.

It was shown >20 yr ago that mice will spontaneously accept renal allografts in the absence of immunosuppression, but the mechanism responsible for this is not understood. We transplanted DBA/2 (H-2(d)) kidneys into nephrectomized C57BL/6 (H-2(b)) mice, and the allografts were spontaneously accepted for >60 days without immunosuppression. In contrast, nonimmunosuppressed cardiac and skin allografts in the same strain combination are rejected within approximately 10 days. The accepted renal allografts have a prominent leukocytic infiltrate, suggesting an ongoing, local immune response. At 60 days post-transplant, the recipients of accepted renal allografts display DBA/2-reactive alloantibodies. They also display DBA/2-reactive delayed-type hypersensitivity responses that are actively counter-regulated by DBA/2-induced TGF-beta production, but not by IL-10 production. These data suggest that a donor-reactive, cell-mediated immune mechanism involving TGF-beta is associated with the spontaneous acceptance of renal allografts in mice.     

Phorbol ester activation of a proteolytic cascade capable of activating latent transforming growth factor-betaL a process initiated by the exocytosis of cathepsin B

12-O-Tetradecanoylphorbol-13-acetate (TPA) suppresses the proliferation of the human breast epithelial cell line MCF10A-Neo by initiating proteolytic processes that activate latent transforming growth factor (TGF)-beta in the serum used to supplement culture medium. Within 1 h of treatment, cultures accumulated an extracellular activity capable of cleaving a substrate for urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA). This activity was inhibited by plasminogen activator inhibitor-1 or antibodies to uPA but not tPA. Pro-uPA activation was preceded by dramatic changes in lysosome trafficking and the extracellular appearance of cathepsin B and beta-hexosaminidase but not cathepsins D or L. Co-treatment of cultures with the cathepsin B inhibitors CA-074 or Z-FA-FMK suppressed the cytostatic effects of TPA and activation of pro-uPA. In the absence of TPA, exogenously added cathepsin B activated pro-uPA and suppressed MCF10A-Neo proliferation. The cytostatic effects of both TPA and cathepsin B were suppressed in cells cultured in medium depleted of plasminogen/plasmin or supplemented with neutralizing TGF-beta antibody. Pretreatment with cycloheximide did not suppress the exocytosis of cathepsin B or the activation of pro-uPA. Hence, TPA activates signaling processes that trigger the exocytosis of a subpopulation of lysosomes/endosomes containing cathepsin B. Subsequently, extracellular cathepsin B initiates a proteolytic cascade involving uPA, plasminogen, and plasmin that activates serum-derived latent TGF-beta.     

Molecular basis of specific inhibition of urokinase plasminogen activator by amiloride

The urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) are very similar serine proteases with the same physiological function, the activation of plasminogen. An increased amount or activity of uPA but not tPA has been detected in human cancers. The PAs are weak proteolytic enzymes, but they activate plasminogen to plasmin, a strong proteolytic enzyme largely responsible for the malignant properties of cancers. It has been shown recently that the administration of uPA inhibitors can reduce tumor size. Inhibitors of uPA could therefore be used as anti-cancer and anti-angiogenesis agents. It has been found that amiloride competitively inhibits the catalytic activity of uPA but not tPA. Modification of this chemical could therefore produce a new class of uPA specific inhibitors and a new class of anti-cancer agents. The X-ray structure of the uPA complex with amiloride is not known. There are structural differences in the specificity pocket of uPA and tPA. However, the potential energy of binding amiloride is lower outside this cavity in the case of tPA. A region responsible for binding amiloride to tPA has been proposed as the loop B93-B101, reached in negatively charged amino acids present in tPA but not uPA.     

A high-affinity receptor for urokinase plasminogen activator on human keratinocytes: characterization and potential modulation during migration.

Low passage cultures of normal human keratinocytes produce several components of the plasminogen activator/plasmin proteolytic cascade, including urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), and two specific inhibitors. Studies here presented demonstrate that these cells also contain a high-affinity (Kd = 3 x 10(-10) M) plasma membrane-binding site for uPA. High molecular weight uPA, either as the single-chain precursor or two-chain activated form, bound to the receptor; however, low molecular weight (33 kD) uPA, tPA, or epidermal growth factor did not compete for binding, demonstrating specificity. Acid treatment, which removed endogenous uPA from the receptor, was required to detect maximal binding (45,000 sites per cell). To investigate the possibility that the uPA receptor on keratinocytes may be involved in epithelial migration during wound repair, cultures were wounded and allowed to migrate into the wounded site. Binding sites for uPA were localized by autoradiographic analysis of 125I-uPA binding as well as by immunocytochemical studies using anti-uPA IgG. With both techniques uPA binding sites were detected selectively on the plasma membrane of cells at the leading edge of the migrating epithelial sheet. This localization pattern suggests that uPA receptor expression on keratinocytes may be coupled to cell migration during cutaneous wounding.     

Mouse ovarian granulosa cells produce urokinase-type plasminogen activator, whereas the corresponding rat cells produce tissue-type plasminogen activator

It is well established that rat ovarian granulosa cells produce tissue plasminogen activator (tPA). The synthesis and secretion of the enzyme are induced by gonadotropins, and correlate well with the time of follicular rupture in vivo. We have found that in contrast, mouse granulosa cells produce a different form of plasminogen activator, the urokinase-type (uPA). As with tPA synthesis in the rat, uPA production by mouse granulosa cells is induced by gonadotropins, dibutyryl cAMP, and prostaglandin E2. However, dexamethasone, a drug which has no effect on tPA synthesis in rat cells inhibits uPA synthesis in the mouse. Results of these determinations made in cell culture were corroborated by examining follicular fluid, which is secreted in vivo predominantly by granulosa cells, from stimulated rat and mouse ovarian follicles. Rat follicular fluid contained only tPA, and mouse follicular fluid only uPA, indicating that in vivo, granulosa cells from the two species are secreting different enzymes. The difference in the type of plasminogen activator produced by the rat and mouse granulosa cells was confirmed at the messenger RNA level. After hormone stimulation, only tPA mRNA was present in rat cells, whereas only uPA mRNA was found in mouse cells. Furthermore, the regulation of uPA levels in mouse cells occurs via transient modulation of steady-state levels of mRNA, a pattern similar to that seen with tPA in rat cells.     

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.     

Insulin-like growth factor-binding protein-5 activates plasminogen by interaction with tissue plasminogen activator, independently of its ability to bind to plasminogen activator inhibitor-1, insulin-like growth factor-I, or heparin

Transgenic mice expressing IGFBP-5 in the mammary gland exhibit increased cell death and plasmin generation. Because IGFBP-5 has been reported to bind to plasminogen activator inhibitor-1 (PAI-1), we determined the effects of this interaction in HC11 cells. PAI-1 prevented plasmin generation from plasminogen and inhibited cleavage of focal adhesions, expression of caspase 3, and cell death. IGFBP-5 could in turn prevent the effects of PAI-1. IGFBP-5 mutants with reduced affinity for IGF-I (N-term) or deficient in heparin binding (HEP- and C-term E and F) were also effective. This was surprising because IGFBP-5 reportedly interacts with PAI-1 via its heparin-binding domain. Biosensor analysis confirmed that, although wild-type IGFBP-5 and N-term both bound to PAI-1, the C-term E had greatly decreased interaction with PAI-1. This suggests that IGFBP-5 does not antagonize the actions of PAI-1 by a direct molecular interaction. In a cell-free system, using tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) to activate plasminogen, PAI-1 inhibited plasmin generation induced by both activators, whereas IGFBP-5 prevented the effects of PAI-1 on tPA but not uPA. Furthermore, we noted that IGFBP-5 activated plasminogen to a greater extent than could be explained solely by inhibition of PAI-1, suggesting that IGFBP-5 could directly activate tPA. Indeed, IGFBP-5 and the C-term E and F were all able to enhance the activity of tPA but not uPA. These data demonstrate that IGFBP-5 can enhance the activity of tPA and that this can result in cell death induced by cleavage of focal adhesions. Thus IGFBP-5 can induce cell death by both sequestering IGF-I and enhancing plasmin generation.     

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.     

Plasminogen activation independent of uPA and tPA maintains wound healing in gene-deficient mice

Simultaneous ablation of the two known activators of plasminogen (Plg), urokinase-type (uPA) and the tissue-type (tPA), results in a substantial delay in skin wound healing. However, wound closure and epidermal re-epithelialization are significantly less impaired in uPA;tPA double-deficient mice than in Plg-deficient mice. Skin wounds in uPA;tPA-deficient mice treated with the broad-spectrum matrix metalloproteinase (MMP) inhibitor galardin (N-[(2R)-2-(hydroxamido-carbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide) eventually heal, whereas skin wounds in galardin-treated Plg-deficient mice do not heal. Furthermore, plasmin is biochemically detectable in wound extracts from uPA;tPA double-deficient mice. In vivo administration of a plasma kallikrein (pKal)-selective form of the serine protease inhibitor ecotin exacerbates the healing impairment of uPA;tPA double-deficient wounds to a degree indistinguishable from that observed in Plg-deficient mice, and completely blocks the activity of pKal, but not uPA and tPA in wound extracts. These findings demonstrate that an additional plasminogen activator provides sufficient plasmin activity to sustain the healing process albeit at decreased speed in the absence of uPA, tPA and galardin-sensitive MMPs and suggest that pKal plays a role in plasmin generation.     

Neutralisation of uPA with a Monoclonal Antibody Reduces Plasmin Formation and Delays Skin Wound Healing in tPA-Deficient Mice

Background

Proteolytic degradation by plasmin and metalloproteinases is essential for epidermal regeneration in skin wound healing. Plasminogen deficient mice have severely delayed wound closure as have mice simultaneously lacking the two plasminogen activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). In contrast, individual genetic deficiencies in either uPA or tPA lead to wound healing kinetics with no or only slightly delayed closure of skin wounds.

Methodology/Principal Findings

To evaluate the therapeutic potential in vivo of a murine neutralizing antibody directed against mouse uPA we investigated the efficacy in skin wound healing of tPA-deficient mice. Systemic administration of the anti-mouse uPA monoclonal antibody, mU1, to tPA-deficient mice caused a dose-dependent delay of skin wound closure almost similar to the delayed kinetics observed in uPA;tPA double-deficient mice. Analysis of wound extracts showed diminished levels of plasmin in the mU1-treated tPA-deficent mice. Immunohistochemistry revealed that fibrin accumulated in the wounds of such mU1-treated tPA-deficent mice and that keratinocyte tongues were aberrant. Together these abnormalities lead to compromised epidermal closure.

Conclusions/Significance

Our findings demonstrate that inhibition of uPA activity with a monoclonal antibody in adult tPA-deficient mice mimics the effect of simultaneous genetic ablation of uPA and tPA. Thus, application of the murine inhibitory mU1 antibody provides a new and highly versatile tool to interfere with uPA-activity in vivo in mouse models of disease.     

Activators and inhibitors of the plasminogen system in Alzheimer's disease

Accumulation and deposition of Aβ is one of the main neuropathological hallmarks of Alzheimer's disease (AD) and impaired Aβ degradation may be one mechanism of accumulation. Plasmin is the key protease of the plasminogen system and can cleave Aβ. Plasmin is activated from plasminogen by tissue plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). The activators are regulated by inhibitors which include plasminogen activator inhibitor-1 (PAI-1) and neuroserpin. Plasmin is also regulated by inhibitors including α2-antiplasmin and α2-macroglobulin. Here, we investigate the mRNA levels of the activators and inhibitors of the plasminogen system and the protein levels of tPA, neuroserpin and α2-antiplasmin in post-mortem AD and control brain tissue. Distribution of the activators and inhibitors in human brain sections was assessed by immunoperoxidase staining. mRNA measurements were made in 20 AD and 20 control brains by real-time PCR. In an expanded cohort of 38 AD and 38 control brains tPA, neuroserpin and α2-antiplasmin protein levels were measured by ELISA. The activators and inhibitors were present mainly in neurons and α2-antiplasmin was also associated with Aβ plaques in AD brain tissue. tPA, uPA, PAI-1 and α2-antiplasmin mRNA were all significantly increased in AD compared to controls, as were tPA and α2-antiplasmin protein, whereas neuroserpin mRNA and protein were significantly reduced. α2-macroglobulin mRNA was not significantly altered in AD. The increases in tPA, uPA, PAI-1 and α2-antiplasmin may counteract each other so that plasmin activity is not significantly altered in AD, but increased tPA may also affect synaptic plasticity, excitotoxic neuronal death and apoptosis.     

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.     

Overexpression of plasminogen activators in the nucleus accumbens enhances cocaine-, amphetamine- and morphine-induced reward and behavioral sensitization

Urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) are extracellular proteases that play a role in synaptic plasticity and remodeling. Psychostimulants induce both tPA and uPA in acute and chronic drug delivery, but cocaine induces preferentially uPA, whereas morphine and amphetamine induce preferentially tPA. Specific doxycline-regulatable lentiviruses expressing these extracellular proteases have been prepared and stereotaxically injected into the nucleus accumbens. We show that tPA-overexpressing animals show greater locomotor activity and behavioral sensitization upon morphine and amphetamine treatments. These effects could be fully suppressed by doxycycline or when tPA had been silenced using small interfering RNAs (siRNAs)-expressing lentiviruses. Furthermore, animals infected with lentiviruses expressing uPA show enhanced conditional place preference for cocaine compared with tPA-overexpressing animals. In contrast, tPA-overexpressing animals when administered amphetamine or morphine showed greater place preference compared with uPA-overexpressing animals. The effects are suppressed when tPA has been silenced using specific siRNAs-expressing vectors. Tissue-type plasminogen activator and uPA possibly induce distinct behaviors, which may be interpreted according to their differential pattern of activation and downstream targets. Taken together, these data add further evidence for a significant function of extracellular proteases tPA and uPA in addiction and suggest a differential role of plasminogen activators in this context.     

Concomitant lack of MMP9 and uPA disturbs physiological tissue remodeling

Urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP9, gelatinase B) have separately been recognized to play important roles in various tissue remodeling processes. In this study, we demonstrate that deficiency for MMP9 in combination with ablation of either uPA- or tissue-type plasminogen activator (tPA)-catalyzed plasminogen activation is critical to accomplish normal gestation in mice. Gestation was also affected by simultaneous lack of MMP9 and the uPA receptor (uPAR). Interestingly, uPA-deficiency additionally exacerbated the effect of MMP9-deficiency on bone growth and an additive effect caused by combined lack in MMP9 and uPA was observed during healing of cutaneous wounds. By comparison, MMP9-deficiency combined with absence of either tPA or uPAR resulted in no significant effect on wound healing, indicating that the role of uPA during wound healing is independent of uPAR, when MMP9 is absent. Notably, compensatory upregulation of uPA activity was seen in wounds from MMP9-deficient mice. Taken together, these studies reveal essential functional dependency between MMP9 and uPA during gestation and tissue repair.     

Elimination of hydrocortisone from the medium enables tissue plasminogen activator gene expression by normal and immortalized nonmalignant human epithelial cells.

Human cervical epithelial cells transfected and immortalized with human papillomavirus type 16 DNA (HCE16/3) can be, like many other epithelial cells, normally grown in medium supplemented with epidermal growth factor, cholera toxin, hydrocortisone, insulin, transferrin, thyroid hormone and serum. We found that hydrocortisone diminished tissue plasminogen activator (tPA) production to an undetectable level. The removal of hydrocortisone increased urokinase plasminogen activator (uPA) activity within 24-48 h and tPA activity within 48-72 h, and converted the cells to a more elongated and fibroblastic phenotype. Upregulation of uPA mRNA was seen as early as at 3 h and of tPA mRNA within 48-72 h. Higher molecular weight forms (97-110 kDa) of plasminogen activators were seen in zymograms, apparently complexed with PAI-1, starting at 6 h both in the presence and absence of hydrocortisone. Immunoprecipitation with a PAI-1 monoclonal antibody confirmed that both uPA and tPA were complexed. We also studied normal diploid human bronchial epithelial cells (NHBE) and NHBE cells transformed with an adeno-12/SV40 hybrid virus (BEAS-2B). In both types of nonmalignant epithelial cells, the removal of hydrocortisone increased uPA activity. The omission of hydrocortisone increased tPA levels significantly in BEAS-2B cell cultures, and in NHBE cell cultures tPA became detectable at 72 h. No PA complexes were seen in these two cell types. We conclude that normal and immortalized nonmalignant epithelial cells produce tPA, but only if hydrocortisone is omitted in the growth medium.     

Antibody-mediated targeting of the urokinase-type plasminogen activator proteolytic function neutralizes fibrinolysis in vivo

Urokinase-type plasminogen activator (uPA) plays a central role in tissue remodeling processes. Most of our understanding of the role of uPA in vivo is derived from studies using gene-targeted uPA-deficient mice. To enable in vivo studies on the specific interference with uPA functionality in mouse models, we have now developed murine monoclonal antibodies (mAbs) directed against murine uPA by immunization of uPA-deficient mice with the recombinant protein. Guided by enzyme-linked immunosorbent assay, Western blotting, surface plasmon resonance, and enzyme kinetic analyses, we have selected two highly potent and inhibitory anti-uPA mAbs (mU1 and mU3). Both mAbs recognize epitopes located on the B-chain of uPA that encompasses the catalytic site. In enzyme activity assays in vitro, mU1 blocked uPA-catalyzed plasminogen activation as well as plasmin-mediated pro-uPA activation, whereas mU3 only was directed against the first of these reactions. We additionally provide evidence that mU1, but not mU3, successfully targets uPA-dependent processes in vivo. Hence, systemic administration of mU1 (i) rescued mice treated with a uPA-activable anthrax protoxin and (ii) impaired uPA-mediated hepatic fibrinolysis in tissue-type plasminogen activator (tPA)-deficient mice, resulting in a phenotype mimicking that of uPA;tPA double deficient mice. Importantly, this is the first report demonstrating specific antagonist-directed targeting of mouse uPA at the enzyme activity level in a normal physiological process in vivo.     

Plasminogen activator expression in F9 teratocarcinoma embryoid bodies and their endoderm derivatives

Plasminogen activators are believed to play an important role in tissue remodeling and cell migration. During mouse embryogenesis, visceral endoderm secretes urokinase-type plasminogen activator (uPA) whereas parietal endoderm secretes tissue-type plasminogen activator (tPA). Visceral endoderm from F9 embryoid bodies can transdifferentiate into parietal endoderm under the appropriate culture conditions. We have examined at the protein and mRNA levels the type of plasminogen activator expressed in whole embryoid bodies, visceral endoderm and its parietal endoderm derivatives. Our experiments show that the visceral endoderm on F9 embryoid bodies synthesizes and secretes substantial amounts of both tPA and uPA. In contrast, the parietal endoderm derived directly from the visceral endoderm secretes dramatically increased levels of tPA and decreases production of uPA to low or below detectable levels. These data support the finding that visceral endoderm can transdifferentiate to parietal endoderm. In addition, this transition provides an excellent model for studying the molecular basis of the coincident down- and upregulation of the two plasminogen activators as well as their potential function during embryogenesis.     

Plasminogen activator inhibitor type-1-deficient mice have an enhanced IFN-gamma response to lipopolysaccharide and staphylococcal enterotoxin B

Plasminogen activator inhibitor type-1 (PAI-1) is a major inhibitor of fibrinolysis by virtue of its capacity to inhibit urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Systemic inflammation is invariably associated with elevated circulating levels of PAI-1, and during human sepsis plasma PAI-1 concentrations predict an unfavorable outcome. Knowledge about the functional role of PAI-1 in a systemic inflammatory response syndrome is highly limited. In this study, we determined the role of endogenous PAI-1 in cytokine release induced by administration of LPS or staphylococcal enterotoxin B (SEB). Both LPS and SEB elicited secretion of PAI-1 into the circulation of normal wild-type (Wt) mice. Relative to Wt mice, PAI-1 gene-deficient (PAI-1(-/-)) mice demonstrated strongly elevated plasma IFN-gamma concentrations after injection of either LPS or SEB. In addition, PAI-1(-/-) splenocytes released more IFN-gamma after incubation with LPS or SEB than Wt splenocytes. Both PAI-1(-/-) CD4+ and CD8+ T cells produced more IFN-gamma upon stimulation with SEB. LPS-induced IFN-gamma release in mice deficient for uPA, the uPA receptor, or tPA was not different from IFN-gamma release in LPS-treated Wt mice. These results identify a novel function of PAI-1 during systemic inflammation, where endogenous PAI-1 serves to inhibit IFN-gamma release by a mechanism that does not depend on its interaction with uPA/uPA receptor or tPA.     

Measuring plasminogen activator inhibitor activity in plasma by two enzymatic assays

We compared two methods that measure plasminogen activator inhibitor (PAI) activity in plasma based on the ability of PAI to inhibit tissue plasminogen activator (tPA) or urokinase (uPA) in order to determine which method most accurately measures plasma PAI activity after stressors, like hemorrhage. Plasma PAI activity was significantly elevated after hemorrhage in both assays. Using standard curves derived from rhPAI-1, we found that the tPA-PAI assay was more sensitive than the uPA-PAI assay. However, we measured a 10-fold difference in PAI activity as measured between assays, suggesting that some endogenous plasma constituents (tPA, uPA, plasminogen or plasmin) may interfere with the accurate determination of PAI activity. Increasing the amount of plasma in each assay led to a progressive increase in PAI activity. However, removing either tPA or plasminogen from the tPA-PAI assay unmasked the presence of some endogenous tPA and plasminogen. Furthermore, increasing plasma volume in either assay increases measured plasma tPA, but not uPA. Finally, plasma tPA is elevated after hemorrhage, whereas plasma uPA is not. These results suggest that endogenous tPA and plasminogen may interfere with the measurement of plasma PAI activity in the tPA-PAI assay after hemorrhage or other stresses. The uPA-PAI assay does not have this confounding problem because endogenous uPA does not interfere with the assay, nor does it rise during hemorrhage.