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Published ahead of print on October 28, 2004, doi:10.1165/rcmb.2004-0289OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 28-34, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0289OC

Imbalanced Plasminogen System in Lymphangioleiomyomatosis

Potential Role of Serum Response Factor

Xiaoning Zhe, Yan Yang and Lucia Schuger

Department of Pathology, Wayne State University, School of Medicine, Detroit, Michigan

Correspondence and requests for reprints should be addressed to Lucia Schuger, M.D., Department of Pathology, Wayne State University, 540 E. Canfield St., Rm. 9248, Detroit, MI 48201. E-mail: lschuger{at}med.wayne.edu


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Pulmonary lymphangioleiomyomatosis (LAM) is characterized by abnormal smooth muscle-like cell (LAM cell) proliferation leading to tissue destruction. We previously demonstrated that serum response factor (SRF), a critical smooth muscle transcription factor, is highly expressed in LAM cells. Here we show that a high SRF level alters the plasminogen (Plg) system. Specifically, overexpression of SRF in human lung fibroblasts upregulated urokinase-type plasminogen activator (uPA) and its substrate Plg, whereas it downregulated plasminogen activator inhibitor (PAI)-1. Because uPA cleaves Plg into plasmin, which activates matrix metalloproteinases (MMP), the end result was an increase in MMP activity. To determine whether uPA, Plg, and PAI-1 were abnormally expressed in LAM in vivo, we immunostained 12 LAM cases. In all cases, the LAM lesions showed stronger immunoreaction for uPA and Plg than the surrounding normal lung parenchyma. On the contrary, PAI-1 was absent in LAM lesions, whereas it was ubiquitous in normal lung parenchyma. Microdissection-based reverse transcriptase/polymerase chain reaction further confirmed upregulation of uPA and Plg and downregulation of PAI-1 message in LAM. Altogether, our findings suggest that the high SRF level seen in LAM contributes to extracellular matrix degradation and progressive LAM cell infiltration of the lung.

Key Words: lung • lymphangioleiomyomatosis • plasminogen • SRF • uPA


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Lymphangioleiomyomatosis (LAM) is characterized by abnormal proliferation of smooth muscle (SM)-like cells (LAM cells) in the lung with formation of LAM cell-rimmed pseudocysts and a high incidence of kidney angiomyolipomas (1, 2). LAM occurs almost exclusively in women and has a mean age of onset in the thirties (3). The most common presenting symptoms are dyspnea and pneumothorax (3, 4). Morphologically, LAM cells resemble immature SM cells and express SM-specific proteins such as SM {alpha}-actin and desmin. Some LAM cells synthesize gp100, a melanocytic marker recognized by antibody HMB45 (5); the presence of such cells is required for definite diagnosis. As a further indication of their dual SM/melanocytic nature, we recently found that essentially all LAM cells immunoreact with antibodies against the melanoma related tetraspanin CD63 (6, 7) and that the majority of LAM cells also immunoreact with PNL2, an antibody against an uncharacterized melanocytic antigen (8, 9).

Although LAM cells lack significant atypia, mitotic activity, or metastatic potential, over time these cells proliferate to destroy the lung parenchyma, creating pseudocysts and leading to progressive loss of pulmonary function and death (10). Furthermore, extrapulmonary extension to mediastinal, cervical, and abdominal lymph nodes has been reported in some cases (11), and recurrent LAM in unilateral transplanted lung has been described (12).

LAM occurs as an isolated disorder or in association with tuberous sclerosis complex (TSC) (13). TSC is an autosomally dominant inherited disease caused by a mutation in the TSC1 or TSC2 tumor suppressor gene, and both have been associated with LAM. The critical role of TSC2 is strongly supported by the findings of Carsillo and colleagues (14), who demonstrated mutations in the TSC2 gene in LAM cells from sporadic LAM cases.

Although the nature of the relationship between LAM and TSC2 remains largely unknown, a growing body of evidence is emerging on the pathway connecting the two. Goncharova and colleagues (15) have shown that tuberin, the product of TSC2, inhibits p70 S6 kinase activation and ribosomal protein S6 phosphorylation, resulting in decreased basal DNA synthesis. Therefore, a lack of tuberin results in enhanced cell proliferation. Moreover, it has been shown in neuroepithelial cells from TSC2 null mice that S6 kinase and S6 phosphorylation activate an mTOR pathway, leading to cell proliferation (16). Proliferation is inhibited by the drug rapamycin, an immunosuppressant that targets mTOR (16). In addition, Noonan and colleagues (17) demonstrated that alteration of CaM signaling through TSC2 may be involved in the pathogenesis of LAM. Other investigators have shown that abnormal expression of cytokines, such as insulin-like growth factor and transforming growth factor-ß1, plays a role in the atypical phenotype of LAM cells (18, 19). Because the etiology of this disease is unclear, the therapeutic approaches have been limited and include the use of progesterone, tamoxifen, and lung transplant for patients with advanced disease (10).

We recently found that LAM lesions express a high level of serum response factor (SRF) compared with the surrounding lung parenchyma, including bronchial and vascular SM. More importantly, high SRF levels produced a pro-proteolytic imbalance in several matrix metalloproteinase (MMP) and their natural inhibitor, namely tissue inhibitor of metalloproteinase (TIMP)-3, that are likely to contribute to the tissue damage and pseudocyst formation seen in LAM (20).

Here we present evidence that SRF also alters the plasminogen (Plg) system in a manner that increases extracellular matrix (ECM) degradation. More specifically, SRF upregulates urokinase-type plasminogen activator (uPA) and Plg, inhibits plasminogen activator inhibitor (PAI)-1, and stimulates plasmin-mediated MMP activation in transfected human lung fibroblasts. In addition, we immunostained 12 LAM cases for uPA, Plg, and PAI-1. In all cases, uPA and Plg were strongly detected in the LAM lesions compared with the surrounding normal lung parenchyma. The opposite was the case for PAI-1, which was present in normal lung parenchyma but absent in LAM lesions. Laser microcapture followed by reverse transcriptase/polymerase chain reaction (RT-PCR) studies confirmed the immunohistochemical findings. These studies therefore suggested that a high SRF level in LAM cells alters the Plg/uPA/PAI-1 equilibrium, resulting in increased MMP activation and ECM degradation.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Cells
Human lung fibroblast cell lines WI-38 and IMR-90 were purchased from American Type Culture Collection (Manassas, VA). Cells were cultured in complete medium composed of Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen), 1.0 mM of sodium pyruvate, 0.1 mM of nonessential amino acids, 1.5 g/l of sodium bicarbonate, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 0.25 µg/ml of fungizone (all from Sigma, St. Louis, MO).

Plasmid Construction and Transient Transfection
SRF-pcDNA3 plasmid was a gift from Dr. Paul Kemp (21). Construction of red fluorescence-tagged SRF-DsRed2 plasmid has been described previously (20). For transient transfection, cells were grown to 70% confluence and transfected using Lipofectamine Plus reagent (Invitrogen) following the manufacturer's instructions. The recombinant plasmids or null vector (used as control) were mixed with the lipofectamine reagent in a 1:3.5 wt/vol proportion, and the cells were transfected for 3 h in OPTI-MEM I medium (Invitrogen). After 3 h of incubation at 37°C, the transfection medium was replaced with complete DMEM, and the cultures were incubated for an additional 18–24 h. The transfection efficiency for the SRF-DsRed2 plasmid construct varied from 25–40%, as indicated by the percentage of cells showing red fluorescence. The SRF plasmid did not allow for determination of transfection efficiency because it expressed untagged protein; however, we assumed that it was similar to or higher than that of the SRF-DsRed2 plasmid because the untagged plasmids are smaller in size.

Immunoblot Analysis
Cells were lysed, and immunoblots were performed as previously described (22, 23). Rabbit polyclonal antibodies to human uPA, PAI-1 (Molecular Innovations Inc., Southfield, MI), and Plg (Santa Cruz Biotechnology Inc., Santa Cruz, CA) were used at a 1:1,000 dilution. Rabbit polyclonal antibody against SRF (Santa Cruz) (24–26) was used at a 1:200 dilution. Primary antibodies were detected with horseradish peroxidase-conjugated secondary antibody diluted 1:3,000 (Bio-Rad Laboratories, Hercules, CA). The bands were visualized by chemiluminescence using a commercial kit (Amersham Life Sciences, Arlington Heights, IL) according to the manufacturer's instructions.

Microdissection of LAM Tissue and RT-PCR Analysis
Twelve fresh-frozen LAM cases were obtained from the NHLBI LAM Registry in compliance with the required regulations and consents. All the cases were obtained during open lung biopsy or lung transplantation, and all were diagnosed with LAM. Serial frozen sections (8 µm thick) from each case were cut and briefly stained with hematoxylin and eosin. One section from each series was immunostained with anti-SM {alpha}-actin antibodies (Boehringer Mannheim Biochemicals Inc., Indianapolis, IN) to confirm the presence of LAM lesion. LAM tissue was then microdissected by using a Leica Laser Microcapture set (Leica Microsystems Inc., Bannockburn, IL). The samples were sorted into two groups: LAM tissue and non-lesion tissue. RNA was isolated from the samples with TRIzol reagent (Invitrogen), and RT-PCR was performed with the GeneAmp RNA PCR kit (PerkinElmer, Foster City, CA) following the manufacturer's instructions. The primers used for PCR were SRF, 5' forward primer, 5'-gacagcagcacagacctcac-3' and 3' reverse primer, 5'-ccctatcacagccatctggt-3'; uPA, 5' forward primer, 5'-tcaccaccaaaatgctgtgt-3' and 3' reverse primer, 5'-aggccattctcttccttggt-3'; PAI-1, 5' forward primer, 5'-ctctctctgccctcaccaac-3' and 3' reverse primer, 5'-gtggagaggctcttggtctg-3'; Plg, 5' forward primer, 5'-gtttgggaatgggaaaggat-3'and 3' reverse primer, 5'-tagcaccagggaccacctac-3'; GAPDH, 5' forward primer, 5'-acccagaagactgtggatgg-3' and 3' reverse primer, 5'-gggtcttactccttggaggc-3'. All amplifications shown here represent the product of22 cycles. GAPDH primer set was used as an internal control. Under the conditions used in these studies, the plateau of internal control and most amplicons was reached at 30 cycles.

Immunohistochemistry
Formalin-fixed, paraffin-embedded tissues from the same cohort described previously were used for immunohistochemical analysis. Five normal lung controls were selected from our pathology archival material. Serial sections (5 µm thick) from each of the cases were immunostained with antibodies against SM {alpha}-actin (Boehringer Mannheim), PNL2 (Dako Cytomation California Inc., Carpinteria, CA), Plg, uPA, and PAI-1. Antibodies were used at a concentration of 4 µg/ml or a 1:50 dilution. For PNL2 immunodetection, antigen retrieval (0.01 M citrate buffer, pH 6.0) was performed before primary antibody incubation. Staining was completed using a commercial peroxidase-anti-peroxidase kit (VECTASTAIN ABC Kit; Vector Laboratories, Burlingame, CA) following the manufacturer's instructions as previously described (22, 27). Human lung fibroblasts transfected with SRF-DsRed2 fusion plasmid were fixed for 5 min in absolute ethanol, immunostained for uPA or PAI-1 antibodies, and exposed to a 1:60 dilution of FITC-conjugated anti-rabbit secondary antibody (Sigma). Controls for these experiments included omission of first antibody and substitution with pre-immune IgG (Sigma).

In Situ Gelatin Degradation Assay
Human lung fibroblasts were plated on a coverslip precoated with 5% DQ gelatin (Molecular Probes, Inc., Eugene, OR) mixed with 10 mg/ml of unlabeled gelatin solution. SRF-DsRed2 transient transfection was performed under the protocol described previously. After transfection, SRF- and vector-transfected cells were divided into two groups: One group received complete DMEM only, and the other was supplemented with 0.5 µM uPA-STOP (as suggested by the manufacturer) or 0.6 µM {alpha}2-antiplasmin (28) (both from American Diagnostica Inc., Stamford, CT). Cells were cultured for an additional 16 h before observation. DQ gelatin is conjugated with quenched fluorescein and yields highly fluorescent peptides upon proteolytic cleavage. The degraded areas were therefore visualized as green fluorescence.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
We previously found that LAM lesions have high SRF levels (20). cDNA array analysis was performed using SRF-transfected lung fibroblasts to identify the potential contribution of SRF to the tissue damage occurring in LAM. The cDNA array data indicated that a high SRF level induced changes in the expression of several ECM-degrading enzymes and their inhibitors (20). Among them, we found that the message for uPA was upregulated, whereas that of PAI-1 was reduced (20). Here we explored these original observations.

The increase in uPA and the decrease in PAI-1 messages observed in the cDNA array studies were confirmed at the protein level by immunoblotting in human lung fibroblasts transiently transfected with SRF (Figure 1A). In additional studies the cells were transfected with a SRF-DsRed2 fusion protein. Figure 1B shows that the cells expressing SRF (red nucleus) produce more uPA (upper picture, arrow) and less PAI-1 (lower picture, arrow) than the nontransfected counterparts (arrowheads). The nuclei have been stained blue with DAPI.



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  		Figure 1. Transient transfection of SRF into human lung fibroblasts upregulates uPA and downregulates PAI-1 expression. (A) Immunoblots showing increase in SRF levels resulting in upregulation of uPA and concomitantly inhibition of PAI-1. EV represents empty vector and UT represents untransfected cells. Ponseau S staining of the membranes is included to indicate equal loading. (B) Transient transfection of SRF-DsRed2 fusion protein into human lung fibroblasts followed by immunostaining using a FITC-conjugated second antibody. The cells expressing SRF (red nucleus) produce more uPA (arrow) and less PAI-1 (arrow) than the nontransfected counterparts (arrowheads). The nuclei have been stained blue with DAPI. Bar: 40 µm.

 
We next determined if our transfection findings represented the in vivo situation. For that, we microdissected LAM lesions from four different cases and used RT-PCR to detect uPA and PAI-1 message levels. As in our transfection studies, LAM lesions showed higher levels of uPA and lower levels of PAI-1 message than the surrounding tissues (labeled as no lesion) (Figure 2A). tPA, the other Plg activator, which is primarily involved in clot dissolution and is expressed by vascular SM, was not present in these cells as indicated by immunohistochemistry (not shown). Immunohistochemical studies also demonstrated high levels of uPA in LAM lesions with essentially no presence of PAI-1 (Figure 2B).



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  		Figure 2. High uPA and low PAI-1 expression in LAM. (A) RT-PCR demonstrating high uPA levels of expression in microcaptured LAM lesions compared with the surrounding uninvolved lung parenchyma (no lesion). The lower panels depict RT-PCR studies demonstrating the opposite pattern for PAI-1, whose expression in microcaptured LAM lesions is downregulated compared with the surrounding uninvolved parenchyma. (B) Immunohistochemistry showing strong positivity for uPA in the LAM lesions (left upper panel, pointed at with black arrowheads) compared with alveoli (#) and normal fibro-connective tissue (normal fibroblasts) (*); reactive epithelial cells are also positive for uPA (white arrowhead). The two small pictures below represent successive sections of the highlighted rectangle in the left upper panel immunostained for SM {alpha}-actin (SMA) and PNL2. The right upper panel shows absence of PAI-1 in LAM lesions (black arrowheads) but PAI-1 presence in epithelial cells (white arrowhead in inset) and blood vessel wall. The two small pictures below represent successive sections of the highlighted rectangle in the right upper panel immunostained with anti-SMA and PNL2 antibodies. Bars: 200 µm in upper panels and 160 µm in small lower panels.

 
We determined whether SRF had an effect on Plg by performing additional transient transfections. These studies indicated that SRF upregulated Plg levels in human lung fibroblasts (Figure 3A). Immunohistochemical studies demonstrated that LAM lesions contain abundant Plg (Figure 3B). Microdissection of LAM lesions followed by RT-PCR confirmed the high level of Plg message compared with the surrounding lung parenchyma (Figure 3C).



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  		Figure 3. SRF stimulates Plg synthesis, which is abundant in LAM lesions. (A) Immunoblot showing that transient transfection with SRF stimulates the synthesis of Plg in human lung fibroblast cells. EV, empty vector; UT, untransfected cells. Ponseau S staining is shown to demonstrate equal loading. (B) Immunostaining for Plg. Notice a more intense reactivity in the LAM lesions compared with the surrounding fibroconnective tissue. The two small pictures below represent successive sections of the highlighted rectangle in the upper figure immunostained with SMA and PNL2. Bars: 200 µm in the upper picture and 180 µm in small lower panels. (C) RT-PCR on tissues microdissected from four cases showing increased Plg expression in LAM lesions compared with surrounding lung parenchyma.

 
Because uPA cleaves Plg into plasmin, which in turn activates MMPs, we studied the ability of cells transfected with SRF to degrade a gelatin substrate (target of active MMP-2 and MMP-9) and found that only SRF-transfected cells showed MMP-2 and/or MMP-9 activity, seen as cells decorated with green fluorescence (Figure 4, arrows). In comparison, the nontransfected cells showed traces to even absent fluorescence, indicating low activity. Furthermore, MMP activity was abolished by uPA-STOP, a synthetic uPA blocker (29) (Figure 4, left lower panel) and by {alpha}2-antiplasmin, the natural inhibitor of plasmin (30) (Figure 4, right lower panel), indicating that the activity was mediated by plasmin cleavage of proMMPs. Cells transfected with vector control had no gelatinase activity (not shown). Our findings showed that SRF alters the Plg system in a way that promotes ECM degradation.



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  		Figure 4. High level of SRF expression activates gelatinases (MMP-2 and/or MMP-9) in human lung fibroblasts. The upper six panels show that cells expressing SRF-DsRed2 fusion protein (red nucleus) degrade gelatin (green areas surrounding cell membranes, arrows) due to activation of MMP-2 and/or MMP-9, whereas nontransfected counterparts produce comparatively much lower levels of gelatinase, shown as trace to no fluorescence. uPA-STOP and {alpha}2-antiplasmin treatments remarkably inhibit gelatinase activity in the SRF-transfected cells (lower two panels, arrowheads). The nuclei have been stained blue with DAPI. Bar: 80 µm.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
LAM is a rare and fatal disease characterized by abnormal proliferation of SM-like cells in the lung, leading to progressive parenchymal destruction and respiratory insufficiency (31). The mechanisms whereby the lung is damaged during the course of this disease are not fully understood. However, there is evidence that increased ECM degradation is part of the pathophysiology of LAM (31–33).

We recently found that LAM lesions express high levels of SRF compared with the surrounding lung parenchyma, including bronchial and vascular SM (20). SRF is a critical muscle-related transcription factor highly expressed in developing SM cells (24) but with low expression in their mature counterparts (25, 26). SRF is a member of the MADs box family of transcription factors (34), binds to the CArG box or CArG box-like motif, is an essential cis-element present in most muscle-specific proteins, and stimulates their transcription (24, 25, 35).

To determine whether high SRF levels might contribute to the progression of LAM, we overexpressed this transcription factor in human lung fibroblasts (20). We found that SRF stimulated MMP-2 and MMP-14 (MT1-MMP), whereas it markedly downregulated one of their inhibitors (TIMP-3). These findings were confirmed in LAM lesions by immunohistochemistry (20). MMP-2 and MMP-14 were previously reported to be upregulated in LAM (32, 33) along with MMP-1, -2, -3, and -9 (33). Therefore, MMP upregulation combined with low TIMP-3 levels might contribute to the connective tissue destruction and cyst formation occurring in LAM.

Here we report that SRF overexpression also induced a pro-proteolytic imbalance in the Plg system that is likely to contribute to the ECM degradation occurring in LAM. The Plg system comprises an inactive proenzyme, Plg, which can be converted to plasmin by either of the two Plg activators, the serine proteases tPA or uPA. Plasmin degrades ECM directly but mainly indirectly by converting latent proMMPs into active MMPs (30). Inhibition of the Plg/MMP system occurs at the level of uPA and tPA by specific Plg activator inhibitors, PAI-1, -2, and -3 (the most important of them being PAI-1) (36). Inhibition can also occur at the level of plasmin by {alpha}2-antiplasmin or at the level of MMPs by TIMPs (30). Although the tPA-mediated pathway is primarily involved in fibrin homeostasis, the uPA-mediated pathway is involved in phenomena such as cell migration and tissue remodeling (30).

Our transient transfection studies indicated that high SRF levels upregulated Plg and uPA and concomitantly inhibited PAI-1. This resulted in plasmin-mediated MMP activation to most of SRF-transfected cells. Some nontransfected cells showed much lower levels of gelatin degradation compared with transfected counterparts, possibly by the action of other proteases such as serine or cysteine, which can degrade gelatin in this artificial situation. We determined that the activation was mainly due to plasmin because when an uPA-specific blocker or the natural plasmin inhibitor {alpha}2-antiplasmin was added to the cultures, MMP activity was markedly reduced. In this assay we used a gelatin substrate; therefore, the data suggest a potential affect of the plasmin system on the activation of MMP-2 and -9, two major gelatinases. Further studies are required to accurately identify if such a link between plasmin and MMP-2 and/or MMP-9 is of significance. An expanding body of evidence has been showing that plasmin directly or indirectly can activate a broad range of MMPs (37). Plasmin directly activates proMMP-1, proMMP-3, proMMP-7, and proMMP-13. Activation of proMMP-2 involves hydrolysis by MT1-MMP, yielding an intermediate that is activated by plasmin. Several active MMPs can further activate other proMMPs, so plasmin plays a critical role in MMP activation and ECM degradation (37).

To the best of our knowledge, the Plg system was never studied in the context of LAM; therefore, we determined whether the changes that we observed in vitro were present in vivo. The alterations in Plg/uPA and PAI-1 were confirmed in 12 LAM cases using a combination of immunohistochemistry and microdissection followed by RT-PCR.

A connection between SRF and the Plg system has not been previously reported. Therefore, the mechanism whereby SRF modulates components of the Plg system has not been studied. The uPA and PAI-1 promoters contain CArG boxes, and therefore their expression could be directly regulated by SRF (38, 39). In this regard, the stimulatory effect of SRF over uPA is easy to understand because, in general, SRF stimulates rather than inhibits gene expression. However, SRF also harbors two inhibitory domains that can inhibit its activation domain, as demonstrated using GAL4-SRF constructs (40). Activation of the inhibitory domains may explain why SRF inhibited PAI-1 expression. Furthermore, it has been shown that high SRF levels may inhibit transcription of certain genes normally stimulated by low amounts of SRF (41). Therefore, PAI-1 might be inhibited by high SRF level, whereas it might be stimulated by a low SRF level. Another possibility to explain the different effects of SRF on uPA and PAI-1 is the existence of at least two classes of SRF target genes based on their relative sensitivity to RhoA-actin and MEK-ERK signaling pathways (42). Thus, uPA may be stimulated through one signaling pathway, whereas PAI-1 may be inhibited through another. All these possibilities are not mutually exclusive and might act in combination.

The plg gene does not have SRF CArG box elements in its 5' flanking region (43); therefore, it is unlikely that SRF could control plg expression by direct binding to the gene. However, plg transcription could be stimulated by SRF through an indirect mechanism. One possibility may involve some of the SRF-activated AP-1 factors, such as c-fos, fos-b, or jun-b (44); these may bind to the AP-1 sites present in the plg promoter region (45) and thereby, through Fos or Jun, SRF may activate plg transcription.

The biologic role of uPA-mediated plasmin proteolysis directly and through MMP has been extensively studied in several organs—mainly the vasculature, where it has been demonstrated that the Plg system plays a significant role in the migration of SM cells and ECM degradation during remodeling. Examples are neointima formation after vascular injury or transplantation and destruction of the media during atherosclerosis (46). In the lung, the Plg system has been studied in the context of fibrin degradation and fibrosis (36) but not in LAM.

In conclusion, our findings seem to indicate that SRF acts broadly in the regulation of the Plg/MMP system and their inhibitors to create a pro-proteolytic imbalance that allows LAM cells to degrade ECM, consequently promoting lung parenchyma destruction and expansion of the lesions.


   Acknowledgments
 
The authors thank the LAM patients for donating their tissues for this study.


   Footnotes
 
This work was supported by NHLBI grants HL-48730 and HL-67100 (L.S.) and by a Fellowship Award from The LAM Foundation (X.Z.).

Conflict of Interest Statement: X.Z. has no declared conflicts of interest; Y.Y. has no declared conflicts of interest; and L.S. has no declared conflicts of interest.

Received in original form September 13, 2004

Received in final form October 19, 2004


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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