help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on August 12, 2004, doi:10.1165/rcmb.2004-0025OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2004-0025OCv1
31/6/672    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lazar, M. H.
Right arrow Articles by Sisson, T. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lazar, M. H.
Right arrow Articles by Sisson, T. H.
American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 672-678, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2004-0025OC

Plasminogen Activator Inhibitor–1 Impairs Alveolar Epithelial Repair by Binding to Vitronectin

Michael H. Lazar, Paul J. Christensen, Ming Du, Bi Yu, Natalya M. Subbotina, Kerstin E. Hanson, Jean M. Hansen, Eric S. White, Richard H. Simon and Thomas H. Sisson

Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical Center; and Veterans Affairs Medical Center, Ann Arbor, Michigan

Address correspondence to: Thomas H. Sisson, M.D., Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical Center, 1150 West Medical Center Drive, 6301 MSRB III, Ann Arbor, MI 48109-0642. E-mail: tsisson{at}umich.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The pathogenesis of pulmonary fibrosis is thought to involve alveolar epithelial injury that, when successfully repaired, can limit subsequent scarring. The plasminogen system participates in this process with the balance between urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) being a critical determinant of the extent of collagen accumulation that follows lung injury. Because the plasminogen system is known to influence the rate of migration of epithelial cells, including keratinocytes and bronchial epithelial cells, we hypothesized that the balance of uPA and PAI-1 would affect the efficiency of alveolar epithelial cell (AEC) wound repair. Using an in vitro model of AEC wounding, we show that the efficiency of repair is adversely affected by a deficiency in uPA or by the exogenous administration of PAI-1. By using PAI-1 variants and AEC from mice transgenically deficient in vitronectin (Vn), we demonstrate that the PAI-1 effect requires its Vn-binding activity. Furthermore, we have found that cell motility is enhanced by the availability of Vn in the matrix and that the AEC-Vn interaction is mediated, in part, by the {alpha}vß1 integrin. The significant effect of uPA and PAI-1 on epithelial repair suggests a mechanism by which the plasminogen system may modulate pulmonary fibrosis.

Abbreviations: Ab, antibody • AEC, alveolar epithelial cell • Dulbecco's modified Eagle's medium, DMEM • IPF, idiopathic pulmonary fibrosis • PAI-1, plasminogen activator inhibitor-1 • PBS, phosphate-buffered saline • SFM, serum-free media • uPA, urokinase-type plasminogen activator • uPA–/–, mice transgenically deficient in uPA • Vn, vitronectin • Vn–/–, mice transgenically deficient in Vn • WT, wild type


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Idiopathic pulmonary fibrosis (IPF) is an insidiously progressive alveolar scarring disorder associated with a high mortality. Current therapies primarily directed at suppressing inflammation have not successfully altered the course of this disease, and new treatment approaches are desperately needed. A better understanding of the pathogenesis of IPF is required to define novel therapeutic targets. Impaired or aberrant wound healing marked by the failure of epithelial cells to resurface the denuded alveolus has been proposed as a critical component in the pathogenesis of IPF (1). The importance of the epithelium is supported by the observation that active fibrosis occurs in areas of the alveolus where pneumocytes have been injured and/or lost (2). Furthermore, growth factors that stimulate epithelial cell migration and proliferation have been shown to reduce fibrosis in animal models, suggesting that improved epithelial regeneration is capable of limiting collagen deposition (3, 4).

Alterations in the plasminogen activation system play a critical role in the development of pulmonary fibrosis. Patients with IPF and other diseases associated with lung scarring demonstrate impaired plasminogen activation in their bronchoalveolar lavage fluid secondary to increased levels of plasminogen activator inhibitor-1 (PAI-1) (5). The bleomycin model of pulmonary fibrosis recapitulates the increased PAI-1 expression and the impairment in plasminogen activation that is characteristic of human fibrotic disorders. Preserving plasminogen activation in the lung through a targeted deletion of the PAI-1 gene, or through enhanced expression of urokinase-type plasminogen activator (uPA), has been shown to dramatically protect mice from bleomycin-induced mortality and lung scarring (6, 7). Conversely, accentuated impairment of plasminogen activation through PAI-1 overexpression leads to exuberant fibrosis after lung injury (6).

The mechanism by which the plasminogen system modulates pulmonary fibrosis remains unclear. However, prior studies have shown this proteolytic cascade to be involved in cutaneous wound healing, keratinocyte migration, and bronchial epithelial cell migration (8–10). In these studies, plasmin-mediated proteolysis was responsible for cutaneous wound repair and bronchial epithelial migration. Plasmin presumably facilitated these processes by allowing cells to digest extracellular matrix barriers and to modify their matrix footholds. The plasminogen system can also influence cell migration through mechanisms independent of plasmin-mediated proteolytic activity. Stefansson and colleagues demonstrated that PAI-1 can inhibit smooth muscle cell migration by interfering with integrin-mediated vitronectin (Vn) binding (9). Furthermore, PAI-1 has been shown to affect the adhesion of a variety of cell types, including HT-1080 fibrosarcoma cells, by accelerating the turnover of matrix-binding proteins, including integrins and the urokinase receptor (11). Extrapolating from these studies and the mounting evidence that impaired alveolar epithelial restoration is a critical component of pulmonary fibrosis, we postulated that the plasminogen system influences lung scarring by altering alveolar epithelial repair. To investigate the role of the plasminogen system in alveolar epithelial wound healing, we have employed an in vitro model (12). In this model, alveolar epithelial cells (AECs) are grown to a confluent monolayer and then mechanically injured. After injury, the rate of repair has been shown to be dependent upon cell migration (13, 14). Because the balance of uPA and PAI-1 is correlated with both pulmonary fibrosis and the rate of wound repair in other tissues, we hypothesized that this balance would influence the efficiency of AEC healing. In this article, we demonstrate that the exogenous administration of PAI-1 or a deficiency of uPA hinders alveolar epithelial wound repair. Interestingly, the ability of PAI-1 to slow AEC migration does not depend on its antiproteolytic inhibition of plasminogen activation, but rather on its capacity to bind Vn. We also demonstrate that the presence of Vn in the wound matrix improves the efficiency of AEC wound repair. When PAI-1 adheres to Vn, it impairs cell motility, at least in part, by interfering with binding of the {alpha}vß1 integrin to this matrix protein.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Media and Reagents
Dulbecco's modified Eagle's medium (DMEM) and DMEM:Ham's F12 (DMEM:F12 1:1) were purchased from Gibco (Carlsbad, CA). Cellgro Complete serum-free media (SFM) was purchased from Mediatech (Herndon, VA). We obtained fetal bovine serum and the peroxidase-labeled goat anti-rabbit immunoglobulin G secondary antibody (Ab) from BioWhittaker (Walkersville, MD). Rabbit anti-integrin {alpha}v subunit polyclonal Ab and rabbit anti-human ß3 polyclonal Ab were acquired from Chemicon International, Inc. (Temecula, CA). Rat anti-mouse CD29 (ß1 integrin) was purchased from Research Diagnostics, Inc. (Flanders, NJ), and isotype-matched rat monoclonal immunoglobulin G2a was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Human multimeric Vn was purchased from Molecular Innovations, Inc. (Southfield, MI); human glu-plasminogen was purchased from American Diagnostica, Inc. (Greenwich, CT). Human plasma fibronectin and sodium orthovanadate were purchased from Sigma-Aldrich (St. Louis, MO). Protease inhibitors were obtained from Calbiochem (San Diego, CA). The DC Protein Assay Kit was obtained from Bio-Rad (Hercules, CA). Finally, the immunoblotting chemiluminescence detection reagent was purchased from Pierce (Rockford, IL).

PAI-1 Variants
PAI-1 Q123, designated PAI-1 (V–P+), is a PAI-1 variant in which the glutamine residue at position 123 is replaced with a lysine. This substitution greatly reduces PAI-1's affinity for Vn binding but does not alter its antiproteolytic activity (15). PAI-1 14–1, designated PAI-1 (V+P+), is a PAI-1 variant that retains all of the antiproteolytic and Vn-binding properties of native PAI-1, but is resistant to spontaneous conversion to the latent form. Both PAI-1 (V–P+) and PAI-1 (V+P+) were purchased from Molecular Innovations, Inc. (Detroit, MI). PAI-1 RR, designated PAI-1 (V+P–), is a PAI-1 variant with two amino acid alterations (Thr333 to Arg and Ala335 to Arg) in the reactive center loop. These amino acid changes do not influence PAI-1's ability to interact with Vn, but impair its proteolytic activity toward urokinase and other serine proteases (16). PAI-1 (V+P–) was a generous gift of Dr. Daniel A. Lawrence (Department of Vascular Biology, Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, MD).

Animals
All animal experiments were performed in accordance with institutional guidelines set forth by the University Committee on the Use and Care of Animals. Mice genetically deficient in PAI-1 (PAI-1–/–) were originally generated by Dr. Peter Carmeliet (University of Leuven, Belgium) and have been backcrossed with C57BL/6 mice for at least eight generations (17). Vn-deficient mice (Vn–/–) were generated by a targeted disruption of the murine Vn gene; these mice have also been backcrossed with C57BL/6 mice for at least eight generations, and were a generous gift of Dr. David Ginsburg (University of Michigan, Ann Arbor, MI) (18). Urokinase-deficient mice (uPA–/–) were originally generated by Dr. Peter Carmeliet, and were purchased from Jackson Laboratories (Bar Harbor, ME) (19). These mice have been backcrossed onto a C57BL6 background. Wild-type (WT) C57BL/6 mice were purchased from Charles River Laboratories, Inc. (Portage, MI).

Fibronectin Coating of 96-Well Plates
Fibronectin from human plasma was dissolved in phosphate-buffered saline (PBS), and 80 µl of a 40 µg/ml solution was placed into the wells of a 96-well plate (Becton, Dickinson and Co., Franklin Lakes, NJ). The plate was incubated for 24 h at 37°C in 5% CO2, after which the fibronectin solution was removed. Thereafter, the wells were washed with fresh PBS before the plating of primary murine AECs.

AEC Purification
Type II AECs were isolated from WT C57BL/6, PAI-1–/– and Vn–/– mice by the method of Corti and colleagues (20) as modified by Moore and Christensen (21). Briefly, the mice were anesthetized, heparinized, and exsanguinated. The pulmonary vasculature was perfused with PBS until the effluent was free of blood. Next, the lungs were filled with 1 ml of dispase followed by 0.45 ml of low-melting-point agarose. Thereafter, the lungs were excised, placed in iced PBS for 2 min, and transferred to 2 ml of dispase for 45 min at room temperature. The airways were then removed and the lungs were minced in DMEM with 0.01% DNase. The mince was passed through sequential nylon mesh filters (100-, 40-, and 25-µm pores) and the resulting cell suspension was centrifuged. After resuspension in DMEM, the cells were incubated with biotinylated anti-CD32 and anti-CD45 antibodies followed by streptavidin-coated magnetic particles. Next, the cell suspension was placed in a magnetic separator, and the remaining suspended cells were incubated overnight in a tissue culture dish. The nonadherent cells were then plated at a density of 2 x 105 cells/well (200 µl of 106 cells/ml) in 96-well plates coated with fibronectin. Cells were maintained in DMEM with penicillin–streptomycin and 10% serum at 37°C in 5% CO2. The final adherent population contained 4% nonepithelial cells at Day 2 in culture by intermediate filament staining.

Preparation of Murine Serum
Murine serum was obtained from anesthetized mice by performing a right ventricular puncture with a 26-gauge needle. Extracted whole blood was placed into a Microtainer-brand serum separator tube (Becton Dickinson and Co., Franklin Lakes, NJ) and centrifuged (1,500 x g) for 10 min at 4°C. The serum was then collected and incubated for 30 min at 56°C to inactivate complement. Serum was either used immediately or frozen at –20°C and thawed just before use.

A549 Cell Wounding Assay
A total of 2 x 105 cells/well (200 µl of 106 cells/ml; American Type Culture Collection, Manassas, VA) were plated in a 96-well plate in DMEM with 10% serum (either murine serum as indicated or fetal bovine serum) and penicillin–streptomycin. The cells were grown to confluence at 37°C in 5% CO2, at which point the medium was changed to DMEM:F12 with 0.1% bovine serum albumin and penicillin–streptomycin (SFM). The cells were then incubated for 24 h. Next, a 1 ml pipette tip was used to scratch the monolayer to create an artificial wound. The injured monolayer was then washed with PBS to remove detached cells, and the cells were incubated in DMEM:F12 with 0.1% bovine serum albumin during wound healing.

Murine AEC Wounding Assay
The protocol for this assay is similar to the A549 wounding assay with the following exceptions: first, serum was not removed from the culture system 24 h before, but rather, at the time of monolayer wounding; second, wounding occurred on the day the cells became confluent; third, the murine AECs were initially plated on a fibronectin matrix. These alterations in the wounding protocol were done to ensure the health of the native murine AEC monolayer during wound repair.

Assessment of Wound Repair
After wounding and washing, the adherent native AECs or A549 cells were incubated in SFM. PAI-1 inhibitors were diluted in this media to a final concentration of 22.5 µg/ml. The anti-{alpha}v Ab and anti-ß3 stock solutions were also diluted in SFM to a final dilution of 1:50. The anti-ß1 was diluted to a final concentration of 25 µg/ml. Because the rate of wound repair varied between experiments, a set of control conditions was always included, and the treatment effect was compared with this control.

The wound areas were photographed with an inverted brightfield microscope (Leica, Wetzlar, Germany) and images were captured with SPOT digital camera RT Slider using SPOT Advanced 3.0.4 image software (Diagnostic Instruments, Inc.). Pictures were taken immediately after wounding (Day 0) and then at 24 and 48 h (Day 1 and Day 2, respectively). Using NIH ImageJ software (National Institutes of Health, Bethesda, MD), the edge of the wound was traced and the area of the wound calculated. The percent healing on Day 1 and Day 2 was determined by dividing the measured wound area by the Day 0 wound area of the same monolayer.

Bleomycin Exposure
Weight-matched groups of Vn-null and WT mice were anesthetized with an intraperitoneal injection of ketamine (0.1 mg/g body weight; Fort Dodge Animal Health, Fort Dodge, IA), and the trachea was exposed by a cervical incision. Bleomycin (Nippon Kayaku Co., Tokyo, Japan) at a dose of 2.5 U/kg (in 50 µl of PBS) was instilled intratracheally using a 27-gauge needle. Control mice received 50 µl of PBS. The animals were killed at 3 wk and their lungs were removed for hydroxyproline analysis. Mice that appeared moribund were killed early and their lungs were included for analysis.

Hydroxyproline Assay
The hydroxyproline content in whole mouse lungs was measured using the technique of Woessner and colleagues with modifications (22). The lung parenchyma from each animal was homogenized in 1.0 ml of PBS. One milliliter of 12 N HCl was added, and the samples were hydrolyzed at 110°C for 24 h. After the samples had cooled, 5 µl were mixed with 5 µl of citrate–acetate buffer (5% citric acid, 1.2% glacial acetic acid, 7.25% sodium acetate, and 3.4% sodium hydroxide) and 100 µl of chloramine-T solution (1.4% chloramine-T, 10% N-propanol, and 80% citrate–acetate buffer) in a 96-well plate. The mixture was incubated for 20 min at room temperature. A total of 100 µl of Ehrlich's solution (2.5 g p-dimethylaminobenzaldehyde added to 9.3 ml of n-propanol and 3.9 ml of 70% perchloric acid) was added to each well, and the plate was incubated at 65°C for 18 min. The absorbance of each sample was measured at 550 nm. Standard curves were generated for each experiment using a known concentration of reagent hydroxyproline. Results were expressed as micrograms of hydroxyproline contained in total lung tissue.

Statistical Analysis
Values are expressed as mean ± SEM. Differences between experimental conditions were analyzed using paired t tests when comparing two conditions. Statistical differences between multiple conditions were assessed using an analysis of variance with post-hoc Newman-Keuls multiple comparison test analysis (GraphPad Prism, GraphPad Software, Inc., San Diego, CA). A P value < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of PAI-1 and uPA on AEC Wound Healing
The level of PAI-1 expression is known to influence the extent of fibrosis after lung injury. We hypothesized that one mechanism by which the plasminogen system modulates lung scarring is through its effects on repair of the alveolar epithelium. To investigate whether PAI-1 can inhibit the rate of alveolar epithelial healing, we added a stable variant, PAI-1 (V+P+), to the culture media of AEC monolayers at the time of wounding. In our initial studies, we used the lung-derived A549 epithelial cell line because it is well adapted to form confluent monolayers in tissue culture, and it expresses relevant components of the plasminogen system, including uPA, PAI-1, and the uPA receptor (23, 24). As shown in Figure 1A, the addition of PAI-1 (V+P+), as we hypothesized, slowed the A549 wound closure rate on both Days 1 and 2 after injury (Figure 1, P < 0.0001). The impaired healing did not appear to result from diminished cell viability because the PAI-1–exposed cells continued to exclude trypan blue (data not shown). In addition, PAI-1 did not bring about cell detachment or changes in cellular morphology as viewed under phase-contrast microscopy. Because A549 cells are a tumor-derived cell line and may exhibit altered migratory behavior when compared with native AECs, we studied the effect of PAI-1 (V+P+) on the rate of wound healing of primary AECs isolated from C57BL/6 mice. As demonstrated in Figure 1B, exogenous PAI-1 also slowed the rate of primary AEC wound repair (P < 0.001).



View larger version (18K):
[in this window]
[in a new window]
  		Figure 1. The effect of PAI-1 on AEC wound healing. (A) A549 cells were grown to confluence and wounded. The rate of wound repair was measured at 24 and 48 h in the presence or absence of PAI-1 (V+P+). The percent closure is defined as the area of the wound in relation to the area of the wound at Day 0. (B) WT AECs were grown to confluence on fibronectin-coated plates in 10% murine serum from WT mice and then wounded. The rate of wound repair was measured in the presence or absence of PAI-1 (V+P+), and the percent closure is defined as the area of the wound at 24 h as compared with the area of the wound at time 0. Error bars represent ± 1 SEM.

 
After discovering that exogenous PAI-1–inhibited AEC migration, we next wanted to investigate whether PAI-1 deficiency would accelerate wound healing. Primary AECs from PAI-1–/– mice were isolated and compared with C57BL/6 AECs with respect to their rate of repair. Of note, prior studies have demonstrated that primary AECs express PAI-1 (23). As shown in Figure 2, an absence of PAI-1 in our culture system enhanced the healing efficiency (P < 0.01). Furthermore, the addition of exogenous PAI-1 (V+P+) to the PAI-1–null cells slowed repair to a rate similar to that of WT cells (P < 0.001). Finally, to determine whether uPA influenced AEC migration, we wounded monolayers derived from mice deficient in this plasminogen activator. Figure 2 demonstrates that these cells migrate less efficiently than WT cells (P < 0.01). Furthermore, in a separate experiment, we found that the exogenous administration of uPA to A459 cells enhanced wound repair (data not shown).



View larger version (31K):
[in this window]
[in a new window]
  		Figure 2. The effect of PAI-1 deficiency on AEC wound healing. WT and PAI-1–null (PAI-1–/–) AECs were grown to confluence on fibronectin-coated plates in 10% murine serum from WT mice and then wounded. The rate of wound repair was measured, and the percent closure is defined as the area of the wound at 24 h as compared with the area of the wound at time 0. Error bars represent ± 1 SEM.

 
Mechanism by Which PAI-1 Inhibits AEC Wound Healing
PAI-1 has the potential to inhibit cell migration through several mechanisms. First, uPA-mediated plasminogen activation has been shown to facilitate cell migration, presumably through the proteolytic degradation of extracellular matrix barriers and footholds (10). Thus, by blocking uPA-mediated proteolysis, PAI-1 could delay AEC wound closure. Second, PAI-1 has been shown to slow smooth muscle cell migration by blocking their ability to bind to Vn (9). To determine the mechanism by which PAI-1 impaired alveolar epithelial wound closure in our in vitro model system, we took advantage of two PAI-1 variants. The first variant, PAI-1 (V–P+) maintains its antiproteolytic activity but demonstrates greatly reduced affinity for Vn when compared with WT PAI-1 (15). The second variant, PAI-1 (V+P–), demonstrates normal binding to Vn, but has no antiproteolytic activity toward uPA or other proteases (16). In our initial experiments, we studied the effects of these variant inhibitors on A549 wound repair. PAI-1 (V+P–) significantly slowed the A549 wound closure rate (P < 0.05), whereas the PAI-1 (V–P+) had no effect (Figure 3A, P = NS). To confirm that these results were not specific to the A549 cell line, we compared the ability of the stable mutant, PAI-1 (V+P+), and the antiproteolytic mutant, PAI-1 (V–P+), to inhibit C57BL/6 AEC wound healing. As demonstrated in Figure 3B, PAI-1 (V+P+), with its intact Vn binding activity, inhibited wound closure (P < 0.05), whereas the PAI-1 (V–P+) had no effect (P = NS). To confirm that the lack of efficacy of PAI-1 (V–P+) was not secondary to an insufficient quantity of plasminogen substrate in our culture system, exogenous plasminogen was added to the media after wounding in both A549 cells and WT murine AECs. This exogenous plasminogen did not influence wound closure rate in either cell type (data not shown). Taken together, these results indicate that the ability of PAI-1 to delay wound closure in our in vitro model requires its Vn-binding properties but not its antiproteolytic activity.



View larger version (37K):
[in this window]
[in a new window]
  		Figure 3. The mechanism of PAI-1 inhibition of AECs. (A) A549 cells were grown to confluence and wounded. The rate of wound repair was measured in absence of exogenous PAI-1 or in the presence of either PAI-1 (V–P+) or PAI-1 (V+P–), and the percent closure is defined as the area of the wound at 24 h compared with the size of the wound at Day 0. (B) WT AECs were grown to confluence on fibronectin-coated plates in 10% murine serum from WT mice and then wounded. The rate of wound repair was measured in the absence of exogenous PAI-1 or in the presence of either PAI-1 (V+P+) or PAI-1 (V–P+), and the percent closure is defined as the area of the wound at 24 h as compared with the area of the wound at time 0. Error bars represent ± 1 SEM.

 
Effect of Vn on the PAI-1 (V+P–)–Mediated Inhibition of Alveolar Cell Healing
The ability of PAI-1 (V+P–) to slow wound repair suggests that AECs migrate more efficiently when bound to Vn. It is also possible that PAI-1 (V+P–) impairs wound healing by disrupting the adherence of AECs to other extracellular matrix supports. To investigate whether the PAI-1–induced slowing of wound closure requires Vn, we isolated cells from Vn–/– mice and grew them to confluence in media with serum that also lacked Vn. We then assessed whether the stable mutant, PAI-1 (V+P+), with its intact Vn binding properties, would impair wound healing in the absence of Vn. Figure 4A demonstrates that PAI-1 (V+P+) had no effect on AEC migration in the absence of Vn (P = NS) and, as expected, the antiproteolytic variant, PAI-1 (V–P+), continued to lack effect (P = NS). Furthermore, to confirm that the presence of Vn in the matrix enhances wound repair, we compared the migration of primary AECs cultured in WT mouse serum to the migration of Vn–/– AECs cultured in mouse serum from Vn–/– mice. As demonstrated in Figure 4B, the absence of Vn during repair significantly hindered the rate of wound closure (P < 0.05). These results confirm the importance of Vn in alveolar epithelial repair processes.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 4. The effect of Vn on PAI-1 (V+P–)–mediated inhibition of AEC healing. (A) Vn-null (Vn–/–) AECs were grown to confluence on fibronectin-coated plates in 10% murine serum from Vn-null mice (Vn–/–) and then wounded. The rate of wound repair was measured in the absence of exogenous PAI-1 or in the presence of either PAI-1 (V–P+) or PAI-1 (V+P+), and the percent closure is defined as the area of the wound at 24 h as compared with the area of the wound at time 0. (B) Vn-null (Vn–/–) and WT murine AECs were grown to confluence on fibronectin-coated plates in 10% murine serum from Vn-null mice (Vn–/–) or 10% serum from WT mice, and then wounded. The rate of wound repair was measured in the absence of exogenous PAI-1, and the percent closure is defined as the area of the wound at 24 h as compared with the area of the wound at time 0. Error bars represent ± 1 SEM.

 
Source of Vn in AEC Wound Repair Model
To better understand how the source of Vn affects our wound assay, we investigated whether serum-derived or epithelial cell–derived Vn was most responsible for accelerating the rate of wound closure. To address this question, we compared the rate of wound repair between WT and Vn–/– primary AECs initially cultured in mouse serum either containing or deficient in Vn. As shown in Figure 5, the AECs isolated from WT mice and grown to confluence in the presence of WT serum migrated significantly faster than the same cells plated in Vn–/– serum (P < 0.05). Vn–/– AECs grown to confluence in the presence of WT serum also migrated more rapidly than when they were plated in serum deficient in Vn. On the other hand, there was no difference between the rates of wound closure of WT and Vn–/– AECs as long as they formed their respective monolayers in the presence of the same type of serum. Both cell types migrate more rapidly when they are grown to confluence in the presence of Vn-containing serum and more slowly when they are grown to confluence in the absence of this matrix protein. Based on these results, AECs appear to depend primarily on serum-derived Vn to support their migration during wound repair.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 5. The effect of the source of Vn on AEC healing. Vn-null (Vn–/–) and WT murine AECs were grown to confluence on fibronectin-coated plates in 10% murine serum from Vn-null mice (Vn–/–), or 10% serum from WT mice, and then wounded. The rate of wound repair was measured in the absence of exogenous PAI-1, and the percent closure is defined as the area of the wound at 24 h as compared with the area of the wound at time 0. Error bars represent ± 1 SEM.

 
Mechanism of Vn Effect on AEC Migration
PAI-1, when bound to Vn, has been shown to impair cell adherence and migration by interfering with the integrin-binding site located near the amino-terminus of this matrix protein. The {alpha}vß3 and {alpha}vß1 integrins, in particular, have affinity for Vn and have been found to modulate smooth muscle cell migration (9, 25) as well as AEC migration (26). We hypothesized that AECs also require integrin binding to Vn for efficient migration. To test this hypothesis, WT murine AECs were grown to confluence in the presence of WT mouse serum and then wounded. The rate of repair was then measured in the presence or absence of an anti–{alpha}v Ab, an anti–ß1 Ab, or an anti–ß3 Ab. As shown in Figure 6A, the presence of anti–{alpha}v and anti–ß1 Ab significantly slowed WT AEC healing (P < 0.04). Conversely, the anti–ß3 Ab had no effect on cell migration (Figure 6A). Because both {alpha}v and ß1 can bind to other matrix proteins (27–29), we next assessed whether the influence of these integrin subunits on cell migration was Vn-specific. Figures 6B and 6C show that the anti–{alpha}v and anti–ß1 Abs do not inhibit epithelial migration in the absence of this matrix protein (P = NS).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 6. The effect of {alpha}v, ß1, and ß3 blockade on AEC wound healing. (A) WT AECs were grown to confluence on fibronectin-coated plates in 10% murine serum from WT mice and then wounded. The rate of wound repair was measured in the absence or presence of an anti–{alpha}V Ab, an anti–ß1 Ab, or an anti–ß3 Ab; the percent closure is defined as the area of the wound at 24 h as compared with the area of the wound at time 0. (B) Vn-null (Vn–/–) AECs were grown to confluence on fibronectin-coated plates in 10% murine serum from Vn-null mice (Vn–/–) and then wounded. The rate of wound repair was measured in the absence or presence of an anti–{alpha}V or an anti–ß3 Ab. The percent closure is defined as the area of the wound at 24 h as compared with the area of the wound at time 0. Error bars represent ± 1 SEM. (C) Vn-null (Vn–/–) AECs were grown to confluence on fibronectin-coated plates in 10% murine serum from Vn-null mice (Vn–/–) and then wounded. The rate of wound repair was measured in the absence or presence of an anti–ß1 Ab. The percent closure is defined as the area of the wound at 24 h as compared with the area of the wound at time 0. Error bars represent ± 1 SEM.

 
Effect of Vn Deficiency on Bleomycin-Induced Pulmonary Fibrosis
Because Vn improved the efficiency of AEC migration in vitro, we postulated that it would influence the degree of repair and the extent of fibrosis after bleomycin-induced lung injury. To investigate this possibility, WT and Vn–/– mice were challenged with either intratracheal bleomycin or PBS. After 3 wk, their lungs were analyzed for collagen content. Using hydroxyproline as a measure of lung collagen, bleomycin induced a significant increase in the lung collagen content of both Vn–/– and WT mice, an effect not seen in those treated with saline alone (Figure 7). The degree of bleomycin-induced lung fibrosis, however, was not different between the Vn–/– and WT animals.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 7. Collagen content after intratracheal bleomycin injection. WT and Vn-null mice were challenge with intratracheal bleomycin (2.5 U/kg in 50 µl of PBS) or PBS. On Day 21, lung collagen content was assessed by measuring the total amount of hydroxyproline. Error bars represent ± 1 SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Successful repair of the alveolar epithelium has been postulated to be a crucial step in minimizing lung fibrosis after injury. The restoration of an injured alveolar epithelium requires both pneumocyte proliferation as well as migration. In this set of experiments, we have defined several critical components of AEC migration during in vitro wound healing. First, we have demonstrated that the balance of uPA and PAI-1 is critical in determining the efficiency of AEC migration. Second, we have found that the effect of PAI-1 on cell motility requires the presence of Vn, and a mutation in the domain of PAI-1 responsible for Vn-binding abrogates its inhibitory effect on wound repair. Third, alveolar epithelial wound healing proceeds more efficiently when cells have access to Vn in their matrix. Finally, we have shown that the efficient migration of AECs on Vn is dependent upon {alpha}v and ß1 integrin subunit binding. Prior studies have found that PAI-1 adheres to the somatomedin B–like amino terminal end of Vn where it can sterically inhibit the integrin recognition site on this extracellular matrix protein (9, 30, 31). Taken together with these prior studies, our results demonstrate that PAI-1 modulates the rate of alveolar epithelial wound repair by affecting cellular binding to Vn through the {alpha}vß1 integrin.

The mechanism by which the plasminogen system mitigates pulmonary fibrosis is poorly understood. As mentioned previously, successful restoration of the alveolar epithelium has been increasingly recognized as an important step in halting the scarring process. PAI-1 deficiency has been shown to significantly protect against bleomycin-induced fibrosis (6), and the absence of PAI-1 clearly accelerates alveolar epithelial wound healing in our in vitro model. PAI-1–deficient mice also demonstrate accelerated cutaneous wound healing (8). Therefore, we speculate that the absence of PAI-1 protects against pulmonary fibrosis by accelerating the migration (and repair) of the alveolar epithelium, whereas increased levels of PAI-1 worsen scarring by delaying or preventing epithelial restoration.

We have demonstrated that the Ab-mediated inhibition of the {alpha}v and ß1 integrin subunits slows AEC migration only in the presence of Vn. As {alpha}vß1 has known binding specificity for Vn, our data suggest that this integrin is important for AEC movement on this matrix protein. Although the {alpha}vß3 integrin has been shown to modulate migration of smooth muscle cells and AECs (9, 26), an anti-ß3 Ab did not slow AEC wound repair in our studies. Further studies are required to determine if other integrins with Vn specificity, including {alpha}vß5, {alpha}vß6, and {alpha}vß8, participate in AEC migration.

Our results demonstrate that the antiproteolytic activity of PAI-1 had no effect on wound repair. However, the extracellular matrices present in the lung after injury are likely much more complex in their composition and architecture than the matrices present in our in vitro model. As such, it is possible that AECs also require uPA-mediated proteolysis to negotiate these more diverse matrices and that the antiproteolytic effect of PAI-1 might significantly impact AEC migration during in vivo wound repair. Indeed, human bronchial epithelial cell migration is significantly slowed by the administration of an anti-uPA Ab that interferes with plasminogen activation (10). Although not tested, the antiproteolytic effects of PAI-1, by also interfering with plasminogen activation, should have had a similar inhibitory effect on bronchial epithelial cell wound repair. On the other hand, the migration of smooth muscle cells (which closely parallel the behavior of primary AECs) along more complex Matrigel matrices was not inhibited by PAI-1 unless these matrices also included Vn (9). When Vn was incorporated into the complex matrices, only the Vn-binding properties of PAI-1 (and not the anti-proteolytic activity) were required to inhibit cell movement.

Because Vn played such an important role in promoting migration of AECs in our in vitro assay, one might postulate that the transgenic deficiency of this matrix protein would worsen in vivo AEC repair and, in turn, exacerbate the degree of fibrosis after lung injury. Contrary to this hypothesis, 3 wk after bleomycin administration, Vn deficient and WT mice accumulated equivalent quantities of lung collagen. When considered further, these results are in fact entirely congruent with our in vitro experiments. In our culture system, the administration of excess PAI-1, by blocking the integrin binding sites on Vn, slowed repair to a rate similar to that observed when Vn was absent from the matrix. In vivo, bleomycin is known to induce excess PAI-1 expression in WT animals at the site of injury, where it can bind to Vn. The accumulation of PAI-1 in the wound matrix would then interfere with AEC access to Vn for the purpose of migration and, in effect, create a wound environment similar to that of Vn-deficiency. Therefore, in both WT and Vn–/– mice, resurfacing of the denuded alveolus will proceed inefficiently because the epithelium is unable to move across a preferred matrix protein. Based on the hypothesis that the degree of fibrosis is, at least in part, determined by the success of epithelial restoration, we predict that WT mice and Vn–/– mice would develop a similar degree of collagen after lung injury.

Whether PAI-1 modulates pulmonary fibrosis entirely through its Vn-binding properties as we have demonstrated in our in vitro model or whether its antiproteolytic activity is also important remains a matter of speculation and requires further study. The fact that mice deficient in plasminogen develop worse bleomycin-induced lung scarring as compared with littermate control animals implies that uPA-mediated proteolysis is also likely to be important in the pathogenesis of lung fibrosis (32). The overexpression of intrapulmonary uPA also limits bleomycin-induced fibrosis, further suggesting a role for the antiproteolytic activity of PAI-1 in vivo (7, 33). On the other hand, the increased expression of uPA in the lung not only increases plasminogen activation, but could also promote epithelial cell migration on Vn by binding PAI-1 and changing its conformation (34, 35). Once bound by uPA, PAI-1 releases from Vn, exposing the integrin binding sites that facilitate cell movement. Thus, uPA overexpression certainly could be limiting pulmonary fibrosis through a Vn-dependent mechanism. Indeed, the addition of exogenous uPA to our in vitro culture system enhanced migration of AECs although its deficiency slowed repair. Taken together with our observation that the PAI-1 (V–P+) variant had no effect on cell migration indicates that uPA is likely accelerating wound repair in our culture system by releasing PAI-1 from the Vn-rich matrix.

In summary, PAI-1 decreases the migration of AECs by blocking their {alpha}vß1-mediated binding to Vn. In turn, the presence of Vn accelerates migration, and PAI-1's inhibitory effects are lost when Vn is absent from the wound matrix. Future studies are required to define whether this in vitro effect of PAI-1 on AEC wound healing dictates the extent of pulmonary fibrosis that follows intratracheal bleomycin instillation.


   Footnotes
 
Conflict of Interest Statement: M.H.L. has no declared conflicts of interest; P.J.C. has no declared conflicts of interest; M.D. has no declared conflicts of interest; B.Y. has no declared conflicts of interest; N.M.S. has no declared conflicts of interest; K.E.H. has no declared conflicts of interest; J.M.H. has no declared conflicts of interest; E.S.W. has no declared conflicts of interest; R.H.S. has no declared conflicts of interest; and T.H.S. has no declared conflicts of interest.

Received in original form January 26, 2004

Received in final form July 21, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Selman, M., T. E. King, and A. Pardo. 2001. Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann. Intern. Med. 134:136–151.[Abstract/Free Full Text]
  2. Katzenstein, A.-L. A., and J. L. Myers. 1998. Idiopathic pulmonary fibrosis: clinical relevance of pathologic classification. Am. J. Respir. Crit. Care Med. 157:1301–1315.[Free Full Text]
  3. Deterding, R. R., A. M. Havill, T. Yano, S. C. Middleton, C. R. Jacoby, J. M. Shannon, W. S. Simonet, and R. J. Mason. 1997. Prevention of bleomycin-induced lung injury in rats by keratinocyte growth factor. Proc. Assoc. Am. Physicians 109:254–268.[Medline]
  4. Dohi, M., T. Hasegawa, K. Yamamoto, and B. C. Marshall. 2000. Hepatocyte growth factor attenuates collagen accumulation in a murine model of pulmonary fibrosis. Am. J. Respir. Crit. Care Med. 162:2302–2307.[Abstract/Free Full Text]
  5. Kotani, I., A. Sato, H. Hayakawa, T. Urano, Y. Takada, and A. Takada. 1995. Increased procoagulant and antifibrinolytic activities in the lungs with idiopathic pulmonary fibrosis. Thromb. Res. 77:493–504.[CrossRef][Medline]
  6. Eitzman, D. T., R. D. McCoy, X. Zheng, W. P. Fay, T. Shen, D. Ginsburg, and R. H. Simon. 1996. 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.[Medline]
  7. Sisson, T. H., K. E. Hanson, N. Subbotina, A. Patwardhan, N. Hattori, and R. H. Simon. 2002. Inducible lung-specific urokinase expression reduces fibrosis and mortality after lung injury in mice. Am. J. Physiol. Lung Cell. Mol. Physiol. 283:L1023–L1032.[Abstract/Free Full Text]
  8. Chan, J. C. Y., D. A. Duszczyszyn, F. J. Castellino, and V. A. Ploplis. 2001. Accelerated skin wound healing in plasminogen activator inhibitor–1–deficient mice. Am. J. Pathol. 159:1681–1688.[Abstract/Free Full Text]
  9. Stefansson, S., and D. A. Lawrence. 1996. The serpin PAI-1 inhibits cell migration by blocking integrin alpha V beta 3 binding to vitronectin. Nature 383:441–443.[CrossRef][Medline]
  10. Legrand, C., M. Polette, J. M. Tournier, S. de Bentzmann, E. Huet, M. Monteau, and P. Birembaut. 2001. uPA/plasmin system–mediated MMP-9 activation is implicated in bronchial epithelial cell migration. Exp. Cell Res. 264:326–336.[CrossRef][Medline]
  11. Czekay, R. P., K. Aertgeerts, S. A. Curriden, and D. J. Loskutoff. 2003. Plasminogen activator inhibitor-1 detaches cells from extracellular matrices by inactivating integrins. J. Cell Biol. 160:781–791.[Abstract/Free Full Text]
  12. Kheradmand, F., H. G. Folkesson, L. Shum, R. Derynk, R. Pytela, and M. A. Matthay. 1994. Transforming growth factor-alpha enhances alveolar epithelial cell repair in a new in vitro model. Am. J. Physiol. 267:L728–L738.
  13. McGuire, J. K., Q. Li, and W. C. Parks. 2003. Matrilysin (matrix metalloproteinase-7) mediates E-cadherin ectodomain shedding in injured lung epithelium. Am. J. Pathol. 162:1831–1843.[Abstract/Free Full Text]
  14. Atabai, K., M. Ishigaki, T. Geiser, I. Ueki, M. A. Matthay, and L. B. Ware. 2002. Keratinocyte growth factor can enhance alveolar epithelial repair by nonmitogenic mechanisms. Am. J. Physiol. Lung Cell. Mol. Physiol. 283:L163–L169.[Abstract/Free Full Text]
  15. Lawrence, D., M. Berkenpas, S. Palaniappan, and D. Ginsburg. 1994. Localization of vitronectin binding domain in plasminogen activator inhibitor-1. J. Biol. Chem. 269:15223–15228.[Abstract/Free Full Text]
  16. Stefansson, S., E. Petitclerc, M. K. Wong, G. A. McMahon, P. C. Brooks, and D. A. Lawrence. 2001. Inhibition of angiogenesis in vivo by plasminogen activator inhibitor-1. J. Biol. Chem. 276:8135–8141.[Abstract/Free Full Text]
  17. Carmeliet, P., L. Kieckens, L. Schoonjans, B. Ream, A. van Nuffelen, G. Prendergast, M. Cole, R. Bronson, D. Collen, and R. C. Mulligan. 1993. Plasminogen activator inhibitor-1 gene–deficient mice: I. Generation by homologous recombination and characterization. J. Clin. Invest. 92:2746–2755.
  18. Zheng, X., T. Saunders, S. Camper, L. Samuelson, and D. Ginsburg. 1995. Vitronectin is not essential for normal mammalian development and fertility. Proc. Natl. Acad. Sci. USA 92:12426–12430.[Abstract/Free Full Text]
  19. Carmeliet, P., L. Schoonjans, L. Kieckens, B. Ream, J. Degen, R. Bronson, R. De Vos, J. J. van den Oord, D. Collen, and R. C. Mulligan. 1994. Physiological consequences of loss of plasminogen activator gene function in mice. Nature 368:419–424.[CrossRef][Medline]
  20. Corti, M., A. R. Brody, and J. H. Harrison. 1996. Isolation and primary culture of murine alveolar type II cells. Am. J. Respir. Cell Mol. Biol. 14:309–315.[Abstract]
  21. Moore, B. B., M. Peters-Golden, P. J. Christensen, V. Lama, W. A. Kuziel, R. Paine, III, and G. B. Toews. 2003. Alveolar epithelial cell inhibition of fibroblast proliferation is regulated by MCP-1/CCR2 and mediated by PGE2. Am. J. Physiol. Lung Cell. Mol. Physiol. 284:L342–L349.[Abstract/Free Full Text]
  22. Woessner, J. F., Jr. 1961. The determination of hydroxyproline in tissue and protein samples containing small proportions of this amino acid. Arch. Biochem. Biophys. 93:440–447.[CrossRef][Medline]
  23. Lund, L. R. 1996. 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.[CrossRef][Medline]
  24. Lieber, M., B. Smith, A. Szakal, W. Nelson-Rees, and G. Todaro. 1976. A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells. Int. J. Cancer 17:62–70.[Medline]
  25. Dufourcq, P., T. Couffinhal, P. Alzieu, D. Daret, C. Moreau, C. Duplaa, and J. Bonnet. 2002. Vitronectin is upregulated after vascular injury and vitronectin blockade prevents neointima formation. Cardiovasc. Res. 53:952–962.[Abstract/Free Full Text]
  26. Kim, H. J., C. A. Henke, S. K. Savik, and D. H. Ingbar. 1997. Integrin mediation of alveolar epithelial cell migration on fibronectin and type I collagen. Am. J. Physiol. 273:L134–L141.
  27. Faccio, R., M. Grano, S. Colucci, A. Z. Zallone, V. Quaranta, and A. J. Pelletier. 1998. Activation of alphav beta3 integrin on human osteoclast-like cells stimulates adhesion and migration in response to osteopontin. Biochem. Biophys. Res. Commun. 249:522–525.[CrossRef][Medline]
  28. Ross, F. P., J. Chappel, J. I. Alvarez, D. Sander, W. T. Butler, M. C. Farach-Carson, K. A. Mintz, P. G. Robey, S. L. Teitelbaum, D. A. Cheresh, R. Faccio, M. Grano, S. Colucci, A. Z. Zallone, V. Quaranta, and A. J. Pelletier. 1993. Interactions between the bone matrix proteins osteopontin and bone sialoprotein and the osteoclast integrin alpha v beta3 potentiate bone resorption J. Biol. Chem. 268:9901–9907.
  29. Petruzzelli, L., M. Takami, and H. D. Humes. 1999. Structure and function of cell adhesion molecules. Am. J. Med. 106:467–476.[CrossRef][Medline]
  30. Schroeck, F., N. Arroyo de Prada, S. Sperl, M. Schmitt, and M. Viktor. 2002. Interaction of plasminogen activator inhibitor type-1 (PAI-1) with vitronectin (Vn): mapping the binding sites on PAI-1 and Vn. Biol Chem 383(7–8):1143–1149.[CrossRef][Medline]
  31. Deng, G., S. A. Curriden, G. Hu, R. P. Czekay, and D. J. Loskutoff. 2001. Plasminogen activator inhibitor-1 regulates cell adhesion by binding to the somatomedin B domain of vitronectin. J. Cell. Physiol. 189:23–33.[CrossRef][Medline]
  32. Swaisgood, C. M., E. L. French, C. Noga, R. H. Simon, and V. A. Ploplis. 2000. The development of bleomycin-induced pulmonary fibrosis in mice deficient for components of the fibrinolytic system. Am. J. Pathol. 157:177–187.[Abstract/Free Full Text]
  33. Sisson, T. H., N. Hattori, Y. Xu, and R. H. Simon. 1999. Treatment of bleomycin-induced pulmonary fibrosis by transfer of urokinase-type plasminogen activator genes. Hum. Gene Ther. 10:2315–2323.[CrossRef][Medline]
  34. Stefansson, S., S. Muhammad, X.-F. Cheng, F. D. Battey, D. K. Strickland, and D. A. Lawrence. 1998. Plasminogen activator inhibitor-1 contains a cryptic high affinity binding site for the low density lipoprotein receptor-related protein. J. Biol. Chem. 273:6358–6366.[Abstract/Free Full Text]
  35. Lawrence, D. A., S. Palaniappan, S. Stefansson, S. T. Olson, A. M. Francis-Chmura, J. D. Shore, and D. Ginsburg. 1997. Characterization of the binding of different conformational forms of plasminogen activator inhibitor-1 to vitronectin: implications for the regulation of pericellular proteolysis. J. Biol. Chem. 272:7676–7680.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
J. E. Adair, V. Stober, M. Sobhany, L. Zhuo, J. D. Roberts, M. Negishi, K. Kimata, and S. Garantziotis
Inter-{alpha}-trypsin Inhibitor Promotes Bronchial Epithelial Repair after Injury through Vitronectin Binding
J. Biol. Chem., June 19, 2009; 284(25): 16922 - 16930.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
Y.-J. Park, G. Liu, E. F. Lorne, X. Zhao, J. Wang, Y. Tsuruta, J. Zmijewski, and E. Abraham
PAI-1 inhibits neutrophil efferocytosis
PNAS, August 19, 2008; 105(33): 11784 - 11789.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. F. Bove, M. Hristova, U. V. Wesley, N. Olson, K. M. Lounsbury, and A. van der Vliet
Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1{alpha}
J. Biol. Chem., June 27, 2008; 283(26): 17919 - 17928.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
I. P. Luttrell, M. Swee, B. Starcher, W. C. Parks, and K. Chitaley
Erectile dysfunction in the type II diabetic db/db mouse: impaired venoocclusion with altered cavernosal vasoreactivity and matrix
Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H2204 - H2211.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
J. C. Horowitz, D. S. Rogers, R. H. Simon, T. H. Sisson, and V. J. Thannickal
Plasminogen Activation Induced Pericellular Fibronectin Proteolysis Promotes Fibroblast Apoptosis
Am. J. Respir. Cell Mol. Biol., January 1, 2008; 38(1): 78 - 87.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
N. Beaufort, D. Leduc, H. Eguchi, K. Mengele, D. Hellmann, T. Masegi, T. Kamimura, S. Yasuoka, F. Fend, M. Chignard, et al.
The human airway trypsin-like protease modulates the urokinase receptor (uPAR, CD87) structure and functions
Am J Physiol Lung Cell Mol Physiol, May 1, 2007; 292(5): L1263 - L1272.
[Abstract] [Full Text] [PDF]

Home page
 Eur Respir JHome page
P. Dahlem, A. P. Bos, J. J. Haitsma, M. J. Schultz, E. K. Wolthuis, J. C. M. Meijers, and B. Lachmann
Mechanical ventilation affects alveolar fibrinolysis in LPS-induced lung injury
Eur. Respir. J., November 1, 2006; 28(5): 992 - 998.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
F. Maquerlot, S. Galiacy, M. Malo, C. Guignabert, D. A. Lawrence, M.-P. d'Ortho, and G. Barlovatz-Meimon
Dual Role for Plasminogen Activator Inhibitor Type 1 as Soluble and as Matricellular Regulator of Epithelial Alveolar Cell Wound Healing
Am. J. Pathol., November 1, 2006; 169(5): 1624 - 1632.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
D. Roosterman, T. Goerge, S. W. Schneider, N. W. Bunnett, and M. Steinhoff
Neuronal control of skin function: the skin as a neuroimmunoendocrine organ.
Physiol Rev, October 1, 2006; 86(4): 1309 - 1379.
[Abstract] [Full Text] [PDF]

Home page
 Proc Am Thorac SocHome page
M. Selman and A. Pardo
Role of epithelial cells in idiopathic pulmonary fibrosis: from innocent targets to serial killers.
Proceedings of the ATS, January 1, 2006; 3(4): 364 - 372.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2004-0025OCv1
31/6/672    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lazar, M. H.
Right arrow Articles by Sisson, T. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lazar, M. H.
Right arrow Articles by Sisson, T. H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2004 American Thoracic Society.