Regulation of plasminogen activator inhibitor-1 and urokinase by hyaluronan fragments in mouse macrophages

Maureen R. Horton1, Mitchell A. Olman2, Clare Bao1, Kimberly E. White2, Augustine M. K. Choi3,4, Beek-Yok Chin3,4, Paul W. Noble3,4, and Charles J. Lowenstein1

1 Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287; 2 Department of Medicine, University of Alabama School of Medicine, Birmingham, Alabama 35294; 3 Department of Internal Medicine, Yale University School of Medicine, New Haven 06520; and 4 Department of Internal Medicine, Veterans Affairs Connecticut Healthcare System, West Haven, Connecticut 06516


    ABSTRACT
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Pulmonary inflammation and fibrosis are characterized by increased turnover and production of the extracellular matrix as well as an impairment of lung fibrinolytic activity. Although fragments of the extracellular matrix component hyaluronan induce macrophage production of inflammatory mediators, the effect of hyaluronan on the fibrinolytic mediators plasminogen activator inhibitor (PAI)-1 and urokinase-type plasminogen activator (uPA) is unknown. This study demonstrates that hyaluronan fragments augment steady-state mRNA, protein, and inhibitory activity of PAI-1 as well as diminish the baseline levels of uPA mRNA and inhibit uPA activity in an alveolar macrophage cell line. Hyaluronan fragments alter macrophage expression of PAI-1 and uPA at the level of gene transcription. Similarly, hyaluronan fragments augment PAI-1 and diminish uPA mRNA levels in freshly isolated inflammatory alveolar macrophages from bleomycin-treated rats. These data suggest that hyaluronan fragments influence alveolar macrophage expression of PAI-1 and uPA and may be a mechanism for regulating fibrinolytic activity during lung inflammation.

extracellular matrix; lung; chronic fibrosis


    INTRODUCTION
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ABSTRACT
INTRODUCTION
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CHRONIC INFLAMMATION AND FIBROSIS are characterized by accumulation of inflammatory cells and mediators, increased turnover and production of the extracellular matrix (ECM), and alterations in the fibrinolytic system (2, 8, 21, 26, 39, 61). Activated macrophages play an essential role in inflammation through the release of a variety of mediators including reactive oxygen and nitrogen species, proteases, chemokines, cytokines, and growth factors (10, 21, 32). Although macrophages produce and secrete important mediators of fibrinolysis, plasminogen activator (PA) inhibitors [plasminogen activator inhibitor (PAI)-1] and PAs [urokinase-type plasminogen activator (uPA)], there is a paucity of information regarding the molecular mechanisms regulating macrophage expression of PAI-1 and uPA in inflammatory states (9, 49, 53, 58, 59, 62).

Recent studies (10, 28, 34, 60) have shown that fragments of ECM components, generated at sites of inflammation, have different biological activities than their high molecular mass (HMW) precursor molecules and may, in fact, play a role in the activation of inflammatory macrophages. For example, fragments of the ECM components collagen and fibronectin, but not the intact molecules, have been shown to have proinflammatory properties (52). Hyaluronan (HA) is a nonsulfated glycosaminoglycan polymer that is a ubiquitously distributed component of the ECM. In its native form, HA exists as a HMW polymer of repeating disaccharide units of (1right-arrow4)-beta -D-glucuronic acid-(1right-arrow3)-N-acetyl-beta -D-glucosamine, which functions in water homeostasis, plasma protein distribution, and matrix structuring in normal tissues (35). At sites of inflammation, HMW HA is fragmented into lower molecular mass (LMW) forms that stimulate inflammatory macrophages to produce important mediators of tissue injury and repair such as cytokines, chemokines, reactive nitrogen species, and growth factors (1, 4, 20, 22, 23, 25, 42).

Under physiological conditions, the fibrinolytic system is tightly regulated by the balance between PAs and PAIs. uPA, one of the two major plasminogen activators, is a serine protease that is secreted by many different cell types including macrophages (59). Plasminogen is cleaved by uPA to generate plasmin, a protease with broad specificity for fibrin and other matrix proteins. The activity of fluid-phase or receptor-bound uPA is specifically and rapidly inhibited by PAI-1 (37). PAI-1 and PAI-2 are two PAIs, serpins, that have been shown to have potent inhibitory activity against PAs (32, 59). Macrophage PAI-1 is largely secreted; it is a potent inhibitor of both uPA and tissue-type plasminogen activator (tPA), and the congenital absence of PAI-1 produces a phenotype with increased bleeding and fibrinolysis (8, 59). PAI-2 is a less potent inhibitor of uPA and single-chain tPA and is a poor inhibitor of two-chain tPA. PAI-2 is largely intracellular in macrophages, and the congenital absence of PAI-2 produces no distinct murine phenotype (14, 53, 62).

In chronic inflammatory states, there is mounting evidence that the balance between PAs and PAIs may be altered, leading to decreased fibrinolysis, excessive fibrin deposition, and, ultimately, tissue fibrosis and organ damage (2, 9, 26, 49). Indeed, imbalances in PAI-1-uPA regulation have been suggested as playing a role in the pathogenesis of chronic lung inflammation, sepsis, and cancer (3, 5, 12, 15, 47, 56). However, the mechanisms governing fibrinolysis in chronic inflammatory states in vivo are unclear, and, in particular, the role of the ECM in inducing uPA and PAI-1 gene expression in macrophages has not been investigated. Our results show that HA fragments induce PAI-1 and inhibit uPA transcription and activity in murine macrophages and thus suggest a novel mechanism for the regulation of fibrinolysis at sites of inflammation.


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Macrophage isolation and culture. Mouse bone marrow-derived (BMD) macrophages were isolated from female C3H/HeJ lipopolysaccharide (LPS)-hyporesponsive mice purchased from the Jackson Laboratory (Bar Harbor, ME) as previously described (46). After harvest, the cells (11 × 106 cells/dish) were cultured for 5 days in DMEM supplemented with 10% heat-inactivated low-LPS fetal bovine serum, 15% L-cell medium, and 1% penicillin-streptomycin-1% glutamine (Biofluids, Rockville, MD) at 37°C under 8% CO2. The mouse alveolar macrophage (AM) cell line MH-S (40) was purchased from the American Type Culture Collection (Manassas, VA). The cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated low-LPS fetal bovine serum and 1% penicillin-streptomycin-1% glutamine (Biofluids) at 37°C under 5% CO2. To exclude the effects of contaminating LPS on experimental conditions, cell stimulation was carried out in the presence of 10 µg/ml of polymyxin B (Calbiochem-Novabiochem, La Jolla, CA).

Chemicals and reagents. Purified HA fragments from human umbilical cords were purchased from ICN Biomedical (Costa Mesa, CA). The HA ICN preparation was free of protein (<2%) and other glycosaminoglycans, with a peak molecular mass of 200,000 Da (43). Cycloheximide (10 µg/ml) and actinomycin D (50 µg/ml) were purchased from Sigma (St. Louis, MO). Protein G Sepharose was purchased from Pharmacia Biotech (Piscataway, NJ), and Promix 35S-labeled methionine and cysteine were purchased from Amersham (Arlington Heights, IL). Stock solutions of reagents were tested for LPS contamination with a Limulus amebocyte assay (Sigma).

Northern blot analysis of mRNA production. RNA was extracted from confluent cell monolayers with 4 M guanidine isothiocyanate and purified by centrifugation through a 5.7 M cesium chloride gradient for 12-18 h at 35,000 rpm as previously described (46). Total RNA (10 µg RNA/lane) was electrophoresed under denaturing conditions through a 1% formaldehyde-containing agarose gel and was transferred to Nytran membranes (Schleicher and Schuell, Keene, NH). RNA was cross-linked to the filter by ultraviolet cross-linking (Stratagene, La Jolla, CA), and the blots were hybridized overnight with 106 counts · min-1 · ml-1 of 32P-labeled cDNA probes labeled with the random-prime method (Amersham). The murine PAI-1 probe used was the EcoR I-Sph 1.1-kb fragment of murine PAI-1, and the murine uPA probe used was the Hind III 1.1-kb fragment of murine uPA (13, 47, 51). After hybridization, the blots were washed in 2× saline-sodium citrate (SSC)-0.1% SDS (once at room temperature for 30 min) and then 0.1× SSC-0.1% SDS (50°C for 20 min twice). Autoradiography was performed at -70°C against Kodak XAR diagnostic film (Eastman Kodak, Rochester, NY). Integrity of RNA was validated by ethidium bromide staining, and differences in RNA loading were documented by analysis of blots for mRNA aldolase signal (36).

Immunoprecipitation of PAI-1. Immunoprecipitation was performed as described by Bonifacino (6). Briefly, the cells were treated with 8 µCi of 35S-Promix with and without HA fragments for 24 h. The samples were cleared of nonspecific antibody binding by treatment with 50 µl/ml of protein G Sepharose (1 h at 4°C). The resultant supernatant was then incubated (overnight at 4°C) with 2 µl of polyclonal antibody specific to PAI-1 (American Diagnostica, Greenwich, CT). The immune complexes were then precipitated with 50 µl of protein G Sepharose (1 h at 4°C). The beads were then washed four times with dilution (10 mM Tris · HCl, pH 8.0, 150 mM NaCl, 0.1% Triton X-100, 0.025% sodium azide, and 0.1% BSA) and wash (50 mM Tris · HCl, pH 6.8) buffers (6). The boiled supernatant was electrophoresed under reducing conditions on a 10% SDS-polyacrylamide gel. The gel was dried (1 h at 80°C), and autoradiography was performed as described in Northern blot analysis of mRNA production.

Direct and reverse fibrin autography. Direct and reverse fibrin autography (PA and PAI-1 assays, respectively) were performed on equal volumes of SDS-PAGE-separated conditioned medium as previously described (16, 48). Briefly, an agarose gel (final concentration 0.83% Sea Plaque; FMC BioProducts, Rockland, ME) containing purified human plasminogen (final concentration 8.3 µg/ml), bovine fibrinogen (final concentration 8 mg/ml; Sigma) and alpha -thrombin (final concentration 0.5 U/ml) was cast and placed over the Triton X-100-washed polyacrylamide gel. The gels were then incubated for 3-6 h (37°C) in a humidified chamber and photographed under indirect light. The transparent (dark) zones in the opaque overlay gel represent PA activity, and the light zones generated in the reverse fibrin gel (uPA 0.05 IU/ml included in overlay gel) represent PAI activity in locations corresponding to their molecular mass. As described, amiloride specifically inhibited the action of a urokinase standard (Ref. 66/46, National Institute for Biological Standards and Controls, London, UK) but did not affect the activity of tPA (58). Anti-tPA IgG (final concentration 10 µg/ml), but not preimmune IgG, specifically inhibited the action of single-chain tPA (Ref. 83/517, National Institute for Biological Standards and Controls) but not that of the urokinase standard (16, 48, 57).

Total PA activity assay. Total PA activity in conditioned medium was measured in triplicate by a plasmin-based chromogenic assay as previously published (48), with minor modifications. The samples were incubated in acetate buffer to destroy anti-plasmin activity followed by incubation (37°C in a humidified chamber for 4-8 h) with plasminogen and the plasmin-sensitive chromogen S-2251 (Chromogenix, Molndal, Sweden) in the presence of CNBr fragments of fibrinogen. In preliminary experiments, amiloride (0.1 mM) was found to inhibit >98% of the activity of a 10 IU/ml urokinase standard (recombinant murine urokinase; American Diagnostica) while reducing the activity of a 10 IU/ml tPA activity standard by only 3%.

Nuclear run-on assay. Nuclei from confluent monolayers of MH-S cells were harvested by scraping in ice-cold PBS and subsequently isolated by centrifugation through a sucrose cushion (17, 24). Nuclei were then incubated (30 min at 30°C) with 1 M dithiothreitol, 20 mM nucleotide triphosphates, and 100 µCi of [32P]UTP (NEN, Boston, MA) in transcription buffer (20 mM Tris · HCl, pH 8.3, 100 mM KCl, 4.5 mM MgCl2, and 20% glycerol). The reaction was stopped by the addition of termination buffer [50 mM Tris · HCl, pH 8.3, 500 mM NaCl, 5 mM EDTA, 200 µg/ml of DNase I (Promega, Madison, WI), and 750 U/ml of RNase inhibitor (Boehringer Mannheim, Indianapolis, IN)]. The nuclei were then incubated with tRNA (Sigma) for 15 min before the addition of 10% SDS, 0.2 M EDTA, and 5 U of proteinase K (Sigma). After a 15-min incubation, RNA was extracted with phenol-chloroform-isoamyl alcohol, precipitated with 20% trichloroacetic acid, washed with 5% trichloroacetic acid-5% pyrophosphate, dissolved, and reprecipitated with 4 M sodium acetate and 100% ethanol (-80°C for 30 min). The purified radiolabeled RNA was washed (once with 70% ethanol), dried with a Speed Vac concentrator (Savant Instruments, Hicksville, NY), and resuspended in 100 µl of diethyl pyrocarbonate-treated water. Normalized samples (equal counts per minute) were allowed to bind to complementary sequences that were prehybridized to Optitran-S membranes (Schleicher and Schuell). Blots were hybridized for 3-4 days and then washed in 2× SSC-0.1% SDS (once for 5 min at room temperature) and in 0.1 × SSC-0.1% SDS (twice for 20 min at 50°C). Band radioactivity was quantified with a phosphorimager (Molecular Dynamics, Sunnyvale, CA).

Bleomycin administration and bronchoalveolar lavage. Bleomycin was administrated to male Harlan Sprague-Dawley rats according to published methods (30). The rats were anesthetized with inhaled isofluorane. After a tracheostomy, 500 µl of sterile normal saline with 1 U of bleomycin sulfate were instilled into the lungs through a 25-gauge needle inserted between the cartilaginous rings of the trachea. Control animals received saline alone. The tracheostomy site was sutured, and the animals were allowed to recover until the time of bronchoalveolar lavage (BAL). The rats were killed with a lethal injection of Pentothal Sodium (Ampro Pharmaceutical, Arcadia, CA) at specified time points after intratracheal instrumentation.

BAL was performed by cannulating the trachea and instilling and retrieving ~50 ml of sterile normal saline in 5-ml aliquots. The entire lavage volume was centrifuged, and the cell pellet was resuspended in RPMI 1640 medium supplemented with fetal bovine serum and antibiotics. A cell differential was performed on the lavage fluid by Wright-Giemsa stain before centrifugation, and repooling of the cells consistently showed at least 95% macrophages. Cell viability was determined with trypan blue exclusion. AMs were purified by adherence to plastic tissue culture dishes for 1 h at 37°C in RPMI 1640 medium without serum or antibiotics under 5% CO2; nonadherent cells were removed by aspiration, and adherent macrophages were washed in sterile PBS. All subsequent experiments were performed in RPMI 1640 medium without serum.


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HA fragments augmented PAI-1 and diminished uPA mRNA levels in mouse macrophages. To investigate the effect of LMW HA fragments on PAI-1 and uPA mRNA levels in macrophages, we stimulated both an AM cell line, MH-S, and primary BMD macrophages from C3H/HeJ mice with HA fragments for varying time periods and performed Northern blot analysis. Both cell types expressed detectable levels of uPA and minimal levels of PAI-1 mRNA under basal conditions (Fig. 1). HA fragments augmented PAI-1 mRNA yet diminished uPA mRNA levels in a time-dependent fashion in both macrophage populations, with a peak induction of PAI-1 mRNA after 3-6 h of HA exposure and a peak diminution of uPA mRNA after 18-24 h of HA stimulation. However, in BMD macrophages, HA fragments inhibited uPA expression after an initial enhancement at 1 h. The effect of HA fragments on PAI-1 and uPA mRNA levels was independent of the presence of serum in the stimulation medium during HA exposure (data not shown). In summary, these data demonstrate that HA fragments increased PAI-1 and decreased uPA steady-state mRNA levels in two different mouse macrophage populations.


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Fig. 1.   Hyaluronan (HA) fragments augmented plasminogen activator (PA) inhibitor (PAI)-1 and diminished urokinase-type plasminogen activator (uPA) mRNA levels in murine macrophages. A and B: Northern analyses of mRNA derived from MH-S cells and bone marrow-derived (BMD) macrophages (BMDM), respectively, stimulated with HA fragments (100 µg/ml) in serum-free medium over time. Aldolase was used to document differences in RNA loading. C and D: quantitative measurements of uPA and PAI-1 mRNA expression, respectively, by MH-S and BMDM. These data are representative of 4 experiments.

HA fragments augmented PAI-1 and diminished uPA mRNA levels in a concentration-dependent and specific fashion in mouse macrophages. To further delineate the effect of HA fragments on PAI-1 and uPA expression, we determined the macrophage response to varying concentrations of HA fragments as well as to other glycosaminoglycans. HA fragments (average molecular mass 200 kDa) increased PAI-1 and diminished uPA mRNA levels in a dose-dependent manner (Fig. 2). PAI-1 expression was induced with as little as 50 µg/ml of HA fragments and was maximal with 200 µg/ml of HA fragments. uPA expression was inhibited by 10 µg/ml of HA fragments, although maximal inhibition required at least 100 µg/ml of HA. Furthermore, neither PAI-1 nor uPA levels were altered by HMW HA (molecular mass 1,000 kDa), disaccharide HA (molecular mass 0.4 kDa), or other glycosaminoglycans such as chondroitin sulfate A or B or heparan sulfate. Thus the effect of HA fragments on PAI-1 and uPA mRNA levels is concentration dependent and specific.


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Fig. 2.   Dose response and specificity of HA fragments for inducing PAI-1 and inhibiting uPA gene expression in MH-S cells as shown by Northern blot analysis of mRNA derived from MH-S cells stimulated with varying doses (1-200 µg/ml) of HA fragments or alternative extracellular matrix (ECM) components in serum-free medium for 6 h. These data are representative of 4 experiments. unstim, Unstimulated; HMW, high molecular mass.

HA fragments induced PAI-1 protein production, secretion and activity but suppressed uPA activity in MH-S cells. To determine whether HA-induced alterations in PAI-1 and uPA mRNA levels resulted in physiological changes in fibrinolysis, we investigated the effect of HA fragments on protein production and fibrinolytic activity. PAI-1 protein production and secretion were determined by immunoprecipitation of cell-conditioned medium from metabolically labeled ([35S]Met or Cys) HA fragment-stimulated MH-S cells with a polyclonal antibody specific to PAI-1 followed by SDS-PAGE and autoradiography. No PAI-1 protein was immunoprecipitated from the conditioned medium from the unstimulated cells, but a single major band corresponding to a PAI-1 protein (~45 kDa) was immunoprecipitated from the conditioned medium from cells stimulated with HA fragments (Fig. 3). Thus HA fragments induced PAI-1 production and pericellular secretion in murine macrophages.


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Fig. 3.   HA fragments augmented PAI-1 protein production and secretion in MH-S cells. MH-S cells were stimulated in serum-containing medium for 24 h with HA fragments (100 µg/ml) in the presence of [35S]methionine or cysteine. Radiolabeled PAI-1 was immunoprecipitated from conditioned cell medium with a polyclonal antibody to PAI-1 and subjected to SDS-PAGE analysis before autoradiography was performed. These data are representative of 3 identical experiments. Nos. at left, molecular mass.

Total PA activity in conditioned cell medium from HA fragment-stimulated MH-S cells was determined with a plasmin-sensitive chromogen-based assay, and relative uPA and PAI-1 activities were determined by enzyme zymography (fibrin autography). As shown in Table 1, HA fragments dramatically reduced total PA activity. The PA activity was attributed to uPA, given that the urokinase-specific inhibitor amiloride (0.1 mM) inhibited >95% of the total PA activity in the samples (data not shown). Thus incubation of the cells with HA diminished the uPA activity in the conditioned medium by 94 (from 24 to 1.3 IU/ml) and 97% (from 383 to 10.5 IU/ml) in the presence and absence of serum, respectively (Table 1). Of note, virtually no PA activity was detected (<0.3 IU/ml, the lowest level of detection of this assay) in serum-free or serum-containing unconditioned medium. To characterize the PA and PAI activities in more detail, fibrin autography was performed. Direct fibrin autography demonstrated a reduction in the PA activity (lytic zone) around 50 kDa in HA fragment-stimulated cells relative to that in unstimulated cells, irrespective of serum exposure (Fig. 4A, top). We identified the PA responsible for the lytic zones as uPA based on its inhibition by amiloride and on its electrophoretic mobility (Fig. 4A, bottom). Concordant with the uPA suppression, inhibitor activity (PAI-1) was present only in the HA fragment or serum-exposed cells (Fig. 4B). Taken together, these studies demonstrate that HA fragments suppressed macrophage pericellular uPA activity and promoted PAI-1 activity.

                              
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Table 1.   Effect of HA on total PA activity in MH-S cells



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Fig. 4.   HA fragments induced PAI-1 activity and inhibited uPA activity in MH-S cells. MH-S cells were stimulated with HA fragments (100 µg/ml) for 24 h in the presence and absence of serum. Aliquots of conditioned medium were subjected to direct and reverse fibrin enzyme zymography. A: uPA activity (lytic zone) in the absence (top) and presence (bottom) of amiloride. B: inhibitor activity (PAI-1). tPA, tissue-type PA. Nos. at left, molecular mass.

New protein synthesis is not required for the induction of PAI-1 but is required for the inhibition of uPA by HA fragments. To examine the mechanism by which HA regulates PAI-1 and uPA, we pretreated MH-S cells with cycloheximide (CHX) for 30 min before stimulation with HA fragments for 6 h. We found that HA fragments in the presence of CHX still augmented PAI-1 steady-state mRNA levels but no longer diminished uPA mRNA levels (Fig. 5). Thus new protein synthesis appears to be necessary for HA fragments to inhibit uPA gene expression but not to induce PAI-1. Of note, CHX alone minimally induced PAI-1 expression as previously described (38).


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Fig. 5.   Effect of cycloheximide (CHX) on PAI-1 and uPA gene expression by HA fragments. MH-S cells were pretreated with CHX (10 µg/ml) for 30 min before stimulation with CHX (10 µg/ml) with and without HA fragments (100 µg/ml) in serum-free medium for 6 h. mRNA was isolated, and Northern blot analysis was performed. These data are representative of 3 identical experiments.

Transcription was required for the induction of PAI-1 and the inhibition of uPA by HA fragments. To determine whether HA fragments were regulating PAI-1 and uPA gene expression at the level of transcription, we performed Northern blot analysis of HA fragment-stimulated MH-S cells in the presence of the mRNA synthesis inhibitor actinomycin D. Actinomycin D added before stimulation of MH-S cells with HA fragments completely blocked the induction of PAI-1 and the inhibition of uPA gene expression at the level of steady-state mRNA (Fig. 6). These results imply that HA fragments do not affect the stability of PAI-1 and uPA mRNAs.


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Fig. 6.   Transcription was required for the induction of PAI-1 and the inhibition of uPA by HA fragments in MH-S cells. Macrophages were pretreated with actinomycin D (50 µg/ml) for 30 min before stimulation with actinomycin D (50 µg/ml) with and without HA fragments (100 µg/ml) in serum-free medium for 9 h. mRNA was isolated, and Northern blot analysis was performed. This blot is representative of 3 identical experiments.

To further examine the direct effect of HA fragments on PAI-1 and uPA gene transcription, we performed nuclear run-on assays in intact nuclei isolated from MH-S cells stimulated with HA for 5 h. HA fragments markedly induced the transcription of PAI-1 and modestly (three- to fourfold) inhibited the transcription of uPA in MH-S cells (Fig. 7). These complementary findings clearly demonstrate that HA fragments regulated PAI-1 and uPA gene expression at the level of transcription and that new protein synthesis was required for uPA transcription but not for PAI-1 transcription.


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Fig. 7.   HA fragments induced PAI-1 and inhibited uPA gene transcription. MH-S cells were stimulated with HA fragments (100 µg/ml) in serum-free medium for 1 h. Nuclei were isolated, and nuclear run-on analysis was performed as described in METHODS. This blot is representative of 2 identical experiments.

HA fragments augmented PAI-1 and diminished uPA mRNA levels in primary AMs from bleomycin-treated rats. To extend these observations to primary inflammatory AMs, we employed an animal model of lung injury and fibrosis. It has been previously shown in the bleomycin model of lung fibrosis that there was an increased accumulation of PAI-1 and HA fragments as well as a decreased activity of uPA (44, 49). Thus bleomycin or saline was intratracheally administered to rats, and primary AMs were isolated after 9 days by BAL with differential adherence. These isolated macrophages were stimulated in vitro with HA (100 µg/ml) for 6 h, and Northern blot analysis was performed. AMs isolated from saline-treated rats had detectable levels of PAI-1 and uPA mRNA at baseline that were not altered by stimulation with HA fragments (Fig. 8). In contrast, HA fragments both induced PAI-1 and diminished uPA mRNA levels in inflammatory AMs from bleomycin-treated rat lungs. Of note, inflammatory AMs from bleomycin-instilled animals expressed lower basal levels of PAI-1 mRNA and higher basal levels of uPA mRNA than AMs from saline-treated animals (Fig. 8). In summary, HA fragments augmented PAI-1 and diminished uPA mRNA levels selectively in primary AMs isolated from inflamed lungs but not from saline-treated lungs.


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Fig. 8.   HA fragments augmented PAI-1 and diminished uPA mRNA levels in inflammatory alveolar macrophages. Alveolar macrophages were isolated by bronchoalveolar lavage from saline-treated control rats and from rats 9 days after intratracheal bleomycin (Bleo) administration. Cells were stimulated with HA (100 µg/ml) in serum-free medium for 4 h, mRNA was isolated, and Northern blot analysis was performed. This blot is representative of 2 identical experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of this study was to examine the effect of LMW ECM fragments on macrophage expression of the modulators of fibrinolysis. Our laboratory (22-25, 41, 42) has previously shown that HA fragments in sizes similar to those generated at sites of inflammation stimulate mouse macrophage expression of numerous cytokines and chemokines. The major finding of this study is that HA fragments, but not native HMW HA, induce transcription and expression of PAI-1 and simultaneously inhibit the transcription and expression of uPA in mouse macrophages. These results identify a novel role for HA fragments as potential regulators of the fibrinolytic system in inflammation.

HA fragments induced PAI-1 and inhibited uPA gene expression in a time-dependent fashion in the AM cell line MH-S as well as in two types of primary macrophages, BMD macrophages and AMs. Furthermore, the decrease in uPA, but not in PAI-1, required new protein synthesis. Although previous investigators (11, 19, 54) have suggested a role for tumor necrosis factor (TNF)-alpha in the regulation of PAI-1 and uPA in macrophages, we found, using macrophages from TNF-alpha knockout mice as well as specific TNF-alpha blocking antibodies, that HA-induced alterations of PAI-1 and uPA steady-state mRNA levels were independent of TNF-alpha (data not shown). These data support a previously unknown mechanism by which matrix fragments may play a role in regulating the fibrinolytic process that occurs in lung injury and repair. Our laboratory (22, 42, 46) has previously shown that HA fragments induce inflammatory gene expression in certain macrophage populations. Our laboratory (22, 42, 46) has also provided evidence to support a role for CD44 in mediating HA fragment-induced gene expression under certain circumstances. However, Horton et al. (25) and others (29) have also provided data that there may be CD44-independent signaling pathways in macrophages as well. Further studies will be required to characterize the role of other potential HA receptors in mediating signal transduction pathways.

The regulation of PAI-1 and uPA by HA fragments appears to be dependent on the state of macrophage activation. Activated and inflammatory macrophages respond to HA fragment stimulation, whereas inactive or quiescent macrophages do not respond. SV-40-transformed MH-S cells, BMD macrophages (which were cultured in colony-stimulating factor-1), and inflammatory AMs from bleomycin-treated rats are all in a state of activation, and all respond to HA fragment stimulation by increasing PAI-1 and decreasing uPA expression. In contrast, noninflammatory AMs from saline-treated rats display baseline levels of PAI-1 and uPA that are unresponsive to HA stimulation. These results are consistent with previously published data from our laboratory (22, 41) showing that HA fragment-induced macrophage gene expression depends on both the origin and state of activation of the macrophages studied. Furthermore, the basal expression of both uPA and PAI-1 and their response to various agonists (phorbol 12-myristate 13-acetate, colony-stimulating factor-1, cholera toxin, interferon-gamma , or LPS) have been shown by other investigators (9, 57) to also be dependent of the level of macrophage activation.

The effect of HA fragments on the AM cell line expression of PAI-1 and uPA occurred in a dose-dependent fashion, was not a general characteristic of glycosaminoglycans, and was specific for LMW HA fragments (200 kDa). Previous investigators (60, 61) have demonstrated that HA oligosaccharides (12-16 disaccharide units, molecular mass 5 kDa) are angiogenic and stimulate endothelial cell proliferation. Furthermore, these HA oligosaccharides promote in vitro invasion of bovine microvascular endothelial cells into a collagen matrix as well as induce gene expression and activity of both uPA and PAI-1 (43, 60, 61). In this report, only the LMW HA fragments (200 kDa) altered macrophage expression of PAI-1 and uPA. Macrophage expression of PAI-1 and uPA was not affected by the HMW HA (1,000 kDa) or by the HA disaccharides (0.4 kDa). We found that the identical HA oligosaccharide preparation, which was previously shown by West and colleagues (60, 61) to induce both PAI-1 and uPA in endothelial cells, did not alter macrophage mRNA levels of PAI-1 or uPA (data not shown). Furthermore, we demonstrated that LMW HA fragments induced macrophage gene expression of PAI-1 and inhibited macrophage expression of uPA, whereas West and colleagues showed that HA oligosaccharides induced both PAI-1 and uPA in endothelial cells. Thus it appears as if the effect of HA fragments is not only dependent on the size of HA but is also cell-type specific.

A similar coordinated reduction in uPA expression or transcription with increases in PAI-1 expression or transcription has been observed in vivo in AMs and in vitro in a murine macrophage cell line (RAW) under conditions of hypoxia (50). Additionally, a previous work (31) on the matrix proteins indicated that the basement membrane protein laminin upregulated uPA expression and fibrinolytic activity in vitro in two murine macrophage cell lines in an integrin (alpha 6-subunit)-dependent manner, whereas collagen, tenascin, and fibronectin were ineffective. Our findings of coordinated induction of PAI-1 expression and inhibition of uPA expression by matrix fragments generated at the site of inflammation adds spatial relevance to the previously described developmental alterations in the responses of these genes. Together, these data suggest that monocyte/macrophage lineage cells respond to an inflammatory stimulus (i.e., cytokines) in a complex manner that is modulated by the underlying level of activation and matrix environment.

In many chronic inflammatory and fibrotic conditions such as adult respiratory distress syndrome, idiopathic pulmonary fibrosis, and sarcoidosis, there are alterations in fibrinolytic expression (2, 3, 26, 27, 49, 54). Bleomycin-induced lung injury is a well-characterized animal model of pulmonary fibrosis that causes an acute alveolitis followed by a fibroproliferative phase (7). In bleomycin-injured lungs, there is both increased PAI-1 and decreased uPA activity as well as increased production and accumulation of HA fragments (in sizes similar to those of our active HA fragments) (7, 44, 45, 49). In fact, mice deficient in PAI-1 expression are relatively resistant to the fibrosing effects of bleomycin (15). Although the mechanisms responsible for the derangement in fibrinolytic activity found in this model are unclear, primary fibroblast-like cells, macrophages, and epithelial cells appear to play a role (18, 47, 55).

Interestingly, AMs isolated from rats 9 days after bleomycin insult have diminished basal levels of PAI-1 and enhanced mRNA levels of uPA compared with AMs from saline-treated control rats. However, on stimulation with HA fragments, the AMs from bleomycin-treated rats have decreased uPA and increased PAI-1 mRNA levels, whereas the AMs from saline-treated animals are unresponsive to HA. Although one might expect that AMs from bleomycin-injured rats, which are theoretically exposed to HA fragments in vivo, have low levels of uPA and high levels of PAI-1 at baseline, we do not find this to be the case, and, in fact, this phenotype is typical of activated macrophages (33, 62). Additionally, these baseline findings may be a reflection of LMW HA clearance (peak HA levels 2-4 days after bleomycin) or of other microenvironmental stimuli modifying HA signaling. Nevertheless, we have demonstrated that HA fragments modify uPA and PAI-1 levels in primary inflammatory AMs.

In conclusion, we have shown that HA fragments of sizes similar to those found in inflammatory tissues alter macrophage uPA and PAI-1 expression as well as pericellular fibrinolytic activity. Identifying the molecular mechanisms by which HA fragments regulate gene expression may lead to new approaches to treat chronic inflammation and fibrosis.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-09614-01 (to M. R. Horton), R01-HL-53615, P50-HL-52315 (both to C. J. Lowenstein), R01-HL-58655 (to M. A. Olman), K11-HL-02880, R01-HL-60539, and 5F32-HL-09614-02 (all to P. W. Noble); the Cora and John H. Davis Foundation (C. J. Lowenstein); the Bernard Bernard Foundation (C. J. Lowenstein); a Merit Award from the Veterans Administration (to M. A. Olman); and the American Lung Association (P. W. Noble).


    FOOTNOTES

Address for reprint requests and other correspondence: P. W. Noble, Yale Univ. School of Medicine, Dept. of Veterans Affairs Connecticut Healthcare System, Pulmonary Section/111A, 950 Campbell Ave., West Haven, CT 06516 (E-mail: paul.noble{at}yale.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5 January 2000; accepted in final form 27 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Balazs, EA, Watson D, Duff IF, and Roseman S. Hyaluronic acid in synovial fluid. I. Molecular parameters of hyaluronic acid in normal and arthritic human fluids. Arthritis Rheum 10: 357-376, 1967[ISI][Medline].

2.   Barazzone, C, Belin D, Piguet PF, Vassalli JD, and Sappino AP. Plasminogen activator inhibitor-1 in acute hyperoxic mouse lung injury. J Clin Invest 98: 2666-2673, 1996[Abstract/Free Full Text].

3.   Bertozzi, P, Astedt B, Zenzius L, Lynch K, LeMaire F, Zapol W, and Chapman HAJ Depressed bronchoalveolar urokinase activity in patients with adult respiratory distress syndrome. N Engl J Med 322: 890-897, 1990[Abstract].

4.   Bitterman, P, Adelberg S, and Crystal R. Mechanisms of pulmonary fibrosis: spontaneous release of the alveolar macrophage-derived growth factor in the interstitial lung disorders. J Clin Invest 72: 1801-1900, 1983[ISI][Medline].

5.   Blasi, F, and Verde P. Urokinase-dependent cell surface proteolysis and cancer. Semin Cancer Biol 1: 117-126, 1990[Medline].

6.   Bonifacino, JS. Immunoprecipitation. In: Current Protocols in Molecular Biology, edited by Ausubel FM.. New York: Wiley, 1991, p. 10.18.1.

7.   Bowden, DH. Unraveling pulmonary fibrosis: the bleomycin model. Lab Invest 50: 487-488, 1984[ISI][Medline].

8.   Carmeliet, P, Stassen JM, Schoonjans L, Ream B, van den Oord JJ, De Mol M, Mulligan RC, and Collen D. Plasminogen activator inhibitor-1 gene-deficient mice. II. Effects on hemostasis, thrombosis, and thrombolysis. J Clin Invest 92: 2756-2760, 1993[ISI][Medline].

9.   Chapman, HA, Jr, Yang XL, Sailor LZ, and Sugarbaker DJ. Developmental expression of plasminogen activator inhibitor type 1 by human alveolar macrophages. Possible role in lung injury. J Immunol 145: 3398-3405, 1990[Abstract/Free Full Text].

10.   Clark, R, Wikner N, Doherty D, and Norris D. Cryptic chemotactic activity of fibronectin for human monocytes resides in the 120-kDa fibroblastic cell-binding fragment. J Biol Chem 263: 12115-12123, 1988[Abstract/Free Full Text].

11.   Colucci, M, Paramo JA, and Collen D. Generation in plasma of a fast-acting inhibitor of plasminogen activator in response to endotoxin stimulation. J Clin Invest 75: 818-824, 1985[ISI][Medline].

12.   Dano, K, Andersen PA, Grondahl-Hansen J, Kristensen P, Nielsen LS, and Skriver L. Plasminogen activators, tissue degradation, and cancer. Adv Cancer Res 44: 139-166, 1985[ISI][Medline].

13.   Degen, SJ, Heckel JL, Reich E, and Degen JL. The murine urokinase-type plasminogen activator gene. Biochemistry 26: 8270-8279, 1987[ISI][Medline].

14.   Dougherty, K, Pearson J, Yang A, Westrick R, and Baker M. The plasminogen activator inhibitor-2 is not required for normal murine development or survival. Proc Natl Acad Sci USA 96: 686-691, 1999[Abstract/Free Full Text].

15.   Eitzman, DT, McCoy RD, Zheng X, Fay WP, Shen T, Ginsburg D, and Simon RH. Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene. J Clin Invest 97: 232-237, 1996[Abstract/Free Full Text].

16.   Erickson, LA, Lawrence DA, and Loskutoff DJ. Reverse fibrin autography: a method to detect and partially characterize protease inhibitors after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal Biochem 137: 454-463, 1984[ISI][Medline].

17.   Greenberg, ME, and Bender TP. Identification of newly transcribed RNA. In: Current Protocols in Molecular Biology, edited by Ausubet FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, and Strull K.. New York: Wiley, 1995, p. 4.10.1-4.10.11.

18.   Gross, T, Simon R, Kelly C, and Sitrin R. Rat alveolar epithelial cells concomitantly express plasminogen activator inhibitor-1 and urokinase. Am J Physiol Lung Cell Mol Physiol 260: L286-L295, 1991[Abstract/Free Full Text].

19.   Gyetko, MR, Shollenberger SB, and Sitrin RG. Urokinase expression in mononuclear phagocytes: cytokine-specific modulation by interferon-gamma and tumor necrosis factor-alpha. J Leukoc Biol 51: 256-263, 1992[Abstract].

20.   Hallgren, R, Eklund A, Engstrom-Laurent B, and Schmekel B. Hyaluronate in bronchoalveolar lavage fluid: a new marker in sarcoidosis reflecting pulmonary disease. Br Med J 290: 1778-1781, 1985[ISI][Medline].

21.   Hance, AJ, and Crystal RG. The connective tissue of lung. Am Rev Respir Dis 112: 657-668, 1975[ISI][Medline].

22.   Hodge-Dufour, J, Noble PW, Horton MR, Bao C, Wysoka M, Burdick MD, Strieter RM, Trinchieri G, and Puré E. Induction of IL-12 and chemokines by hyaluronan requires adhesion-dependent priming of resident but not elicited macrophages. J Immunol 15: 2492-2500, 1997.

23.   Horton, MR, Burdick MD, Strieter RM, Bao C, and Noble PW. Regulation of hyaluronan-induced chemokine gene expression by IL-10 and IFN-gamma in mouse macrophages. J Immunol 160: 3023-3030, 1998[Abstract/Free Full Text].

24.   Horton, MR, McKee CM, Bao C, Liao F, Farber JM, Hodge-DuFour J, Pure E, Oliver BL, Wright TM, and Noble PW. Hyaluronan fragments synergize with interferon-gamma to induce the C-X-C chemokines mig and interferon-inducible protein-10 in mouse macrophages. J Biol Chem 52: 35088-35094, 1998.

25.   Horton, MR, Shapiro S, Bao C, Lowenstein CJ, and Noble PW. Induction and regulation of macrophage metalloelastase by hyaluronan fragments in mouse macrophages. J Immunol 162: 4171-4176, 1999[Abstract/Free Full Text].

26.   Idell, S, James KK, Levin EG, Schwartz BS, Manchanda N, Maunder RJ, Martin TR, McLarty J, and Fair DS. Local abnormalities in coagulation and fibrinolytic pathways predispose to alveolar fibrin deposition in the adult respiratory distress syndrome. J Clin Invest 84: 695-705, 1989[ISI][Medline].

27.   Idell, S, Koenig KB, Fair DS, Martin TR, McLarty J, and Maunder RJ. Serial abnormalities of fibrin turnover in evolving adult respiratory distress syndrome. Am J Physiol Lung Cell Mol Physiol 261: L240-L248, 1991[Abstract/Free Full Text].

28.   Juliano, RL, and Haskill S. Signal transduction from the extracellular matrix. J Cell Biol 120: 577-585, 1993[ISI][Medline].

29.   Khaldoyanidi, S, Moll J, Karakhanova S, Herrlich P, and Ponta H. Hyaluronate-enhanced hematopoiesis: two different receptors trigger the release of interleukin-1beta and interleukin-6 from bone marrow macrophages. Blood 94: 940-949, 1999[Abstract/Free Full Text].

30.   Khalil, N, Berexnay O, Sporn M, and Greenberg AH. Macrophage production of transforming growth factor beta and fibroblast collagen synthesis in chronic pulmonary inflammation. J Exp Med 170: 727-737, 1989[Abstract].

31.   Khan, K, and Falcone D. Role of laminin in matrix induction of macrophage urokinase-type plasminogen activator and 92-kDa metalloproteinase expression. J Biol Chem 272: 8270-8275, 1997[Abstract/Free Full Text].

32.   Krulthof, E, Baker E, Ferrante A, and Antalis T. Biological and clinical aspects of plasminogen activator inhibitor type 2. Blood 86: 4007-4024, 1995[Free Full Text].

33.   Kung, S, and Law H. Modulation of the plasminogen activation system in murine macrophages. Biochim Biophys Acta 1176: 113-122, 1993[ISI][Medline].

34.   Laskin, D, Soltys R, Berg R, and Riley D. Activation of alveolar macrophages by native and synthetic collagen-like polypeptides. Am J Respir Cell Mol Biol 10: 58-64, 1994[Abstract].

35.   Laurent, T, and Fraser J. Hyaluronan. FASEB J 6: 2397-2404, 1992[Abstract/Free Full Text].

36.   Lokuta, M, Maher J, Noe K, Pitha O, Shin M, and Shin H. Mechanisms of murine RANTES chemokine gene induction by Newcastle disease virus. J Biol Chem 271: 13731-13738, 1996[Abstract/Free Full Text].

37.   Loskutoff, DJ, Sawdey M, and Mimuro J. Type 1 plasminogen activator inhibitor. Prog Hemost Thromb 9: 87-115, 1989[ISI][Medline].

38.   Lund, LR. Expression of urokinase-type plasminogen activator, its receptor and type-1 plasminogen activator inhibitor is differently regulated by inhibitors of protein synthesis in human cancer cell lines. FEBS Lett 383: 139-144, 1996[ISI][Medline].

39.   Martinet, Y, Rom WN, Grotendorst GR, Martin GR, and Crystal RG. Exaggerated spontaneous release of platelet-derived growth factor by alveolar macrophages from patients with idiopathic pulmonary fibrosis. N Engl J Med 317: 202-209, 1987[Abstract].

40.   Mbawuike, I, and Herscowitz H. MH-S, a murine alveolar macrophage cell line: morphological, cytochemical, and functional characteristics. J Leukoc Biol 46: 119-127, 1989[Abstract].

41.   McKee, C, Lowenstein C, Horton M, Wu J, Bao C, Chin B, Choi A, and Noble P. Hyaluronan fragments induce nitric oxide in murine macrophages through an NF-kappa B-dependent mechanism. J Biol Chem 272: 8013-8018, 1997[Abstract/Free Full Text].

42.   McKee, C, Penno M, Cowman M, Burdick M, Strieter R, Bao C, and Noble P. Hyaluronan (HA) fragments induce chemokine gene expression in alveolar macrophages. The role of HA size and CD44. J Clin Invest 98: 2403-2413, 1996[Abstract/Free Full Text].

43.   Montesano, R, Kumar S, Orci L, and Pepper MS. Synergistic effect of hyaluronan oligosaccharides and vascular endothelial growth factor on angiogenesis in vitro. Lab Invest 75: 249-262, 1996[ISI][Medline].

44.   Nettelbladt, O, and Hallgren R. Hyaluronan (hyaluronic acid) in bronchoalveolar lavage fluid during the development of bleomycin-induced alveolitis in the rat. Am Rev Respir Dis 140: 1028-1032, 1989[ISI][Medline].

45.   Nettelbladt, O, Scheynius A, Bergh J, Tengblad A, and Hallgren R. Alveolar accumulation of hyaluronan and alveolar cellular response in bleomycin-induced alveolitis. Eur Respir J 4: 407-414, 1991[Abstract].

46.   Noble, P, Lake F, Henson P, and Riches D. Hyaluronate activation of CD44 induces insulin-like growth factor-1 expression by a tumor necrosis factor-alpha -dependent mechanism in murine macrophages. J Clin Invest 91: 2368-2377, 1993[ISI][Medline].

47.   Olman, MA, Mackman N, Gladson CL, Moser KM, and Loskutoff DJ. Changes in procoagulant and fibrinolytic gene expression during bleomycin-induced lung injury in the mouse. J Clin Invest 96: 1621-1630, 1995[ISI][Medline].

48.   Olman, MA, Marsh JJ, Lang IM, Moser KM, Binder BR, and Schleef RR. Endogenous fibrinolytic system in chronic large-vessel thromboembolic pulmonary hypertension. Circulation 86: 1291-1298, 1992[Abstract].

49.   Olman, MA, Simmons WL, Pollman DJ, Loftis AY, Bini A, Miller EJ, Fuller GM, and Rivera KE. Polymerization of fibrinogen in murine bleomycin-induced lung injury. Am J Physiol Lung Cell Mol Physiol 271: L519-L526, 1996[Abstract/Free Full Text].

50.   Pinsky, D, Liao H, Lawson C, Yan S, Chen J, Carmeliet P, Loskutoff D, and Stern D. Coordinated induction of plasminogen activator inhibitor-1 (PAI-1) and inhibition of plasminogen activator gene expression by hypoxia promotes pulmonary vascular fibrin deposition. J Clin Invest 102: 919-928, 1998[Abstract/Free Full Text].

51.   Prendergast, G, Diamond L, Dahl D, and Cole M. The c-myc-regulated gene mrl encodes plasminogen activator inhibitor 1. Mol Cell Biol 10: 1265-1269, 1990[ISI][Medline].

52.   Rennard, SI, Hunninghake PB, Bitterman PB, and Crystal RG. Production of fibronectin by the human alveolar macrophage: mechanism for the recruitment of fibroblasts to sites of tissue injury in interstitial lung diseases. Proc Natl Acad Sci USA 78: 7147-7151, 1981[Abstract].

53.   Ritchie, H, Jamieson A, and Booth N. Regulation, location and activity of plasminogen activator inhibitor 2 (PAI-2) in peripheral blood monocytes, macrophages and foam cells. Thromb Haemost 77: 1168-1173, 1997[ISI][Medline].

54.   Sawdey, MS, and Loskutoff DJ. Regulation of murine type 1 plasminogen activator inhibitor gene expression in vivo. Tissue specificity and induction by lipopolysaccharide, tumor necrosis factor-alpha, and transforming growth factor-beta. J Clin Invest 88: 1346-1353, 1991[ISI][Medline].

55.   Simon, R, and Paine R, III. Participation of pulmonary alveolar epithelial cells in lung inflammation. J Lab Clin Med 126: 108-118, 1995[ISI][Medline].

56.   Suffredini, AF, Harpel PC, and Parrillo JE. Promotion and subsequent inhibition of plasminogen activation after administration of intravenous endotoxin to normal subjects. N Engl J Med 320: 1165-1172, 1989[Abstract].

57.   Vassalli, JD, and Belin D. Amiloride selectively inhibits the urokinase-like plasminogen activator. FEBS Lett 214: 87-91, 1987[ISI][Medline].

58.   Vassalli, JD, Dayer JM, Wohlwend A, and Belin D. Concomitant secretion of prourokinase and of a plasminogen activator-specific inhibitor by cultured human monocytes-macrophages. J Exp Med 159: 1653-1668, 1984[Abstract].

59.   Vassalli, JD, Sappino AP, and Belin D. The plasminogen activator/plasmin system. J Clin Invest 88: 1067-1072, 1991[ISI][Medline].

60.   West, D, Hampson I, Arnold F, and Kumar S. Angiogenesis induced by degradation products of hyaluronic acid. Science 228: 1324-1326, 1985[ISI][Medline].

61.   West, DC, and Kumar S. The effect of hyaluronate and its oligosaccharides on endothelial cell proliferation and monolayer integrity. Exp Cell Res 183: 179-196, 1989[ISI][Medline].

62.   Wohlwend, A, Belin D, and Vassalli J. Plasminogen activator-specific inhibitors in mouse macrophages: in vivo and in vitro modulation of their synthesis and secretion. J Immunol 139: 1278-1284, 1987[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 279(4):L707-L715