help button home button
AJRCCM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on May 25, 2006, doi:10.1164/rccm.200510-1648OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200510-1648OCv1
174/5/557    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 Google Scholar
Google Scholar
Right arrow Articles by Kelly, M. M.
Right arrow Articles by Gauldie, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kelly, M. M.
Right arrow Articles by Gauldie, J.
American Journal of Respiratory and Critical Care Medicine Vol 174. pp. 557-565, (2006)
© 2006 American Thoracic Society
doi: 10.1164/rccm.200510-1648OC

Original Article

Cell-specific Gene Expression in Patients with Usual Interstitial Pneumonia

Margaret M. Kelly, Richard Leigh, Sarah E. Gilpin, Elaine Cheng, Gail E. M. Martin, Katherine Radford, Gerard Cox and Jack Gauldie

Department of Pathology and Molecular Medicine, Centre for Gene Therapeutics, and Firestone Institute for Respiratory Health and Department of Medicine, St. Joseph's Healthcare–McMaster University, Hamilton, Ontario, Canada

Correspondence and requests for reprints should be addressed to Dr. Jack Gauldie, Ph.D., F.R.S.C., Department of Pathology and Molecular Medicine, Centre for Gene Therapeutics, MDCL-4017, McMaster University, 1200 Main Street West, Hamilton, ON, Canada L8N 3Z5. E-mail: gauldie{at}mcmaster.ca


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Usual interstitial pneumonia (UIP) is characterized by extracellular matrix deposition and the development of pulmonary fibrosis. Fibroblastic foci found in the lung are believed to represent an early stage in the evolution of this disease.

Objectives: To compare gene expression profiles in different components of lung tissue (fibroblastic foci, adjacent epithelium, and areas of type 2 pneumocyte hyperplasia) from patients with UIP, and contrast these profiles to distal, uninvolved (control) alveolar tissue from patients undergoing lung resection for cancer.

Methods: Lung resection tissue (UIP, n = 11; controls, n = 11) was snap-frozen for subsequent laser capture microdissection, followed by mRNA extraction, linear amplification, and quantitative real-time polymerase chain reaction.

Results: In patients with UIP, tissue inhibitor of matrix metalloprotease-1 and matrix metalloprotease (MMP)-2 gene expression was up-regulated within the fibroblastic foci compared with the overlying epithelium (p = 0.03, p = 0.02), and to control alveoli (p = 0.001, p = 0.04), respectively. MMP-9 and MMP-7, as well as osteopontin, were up-regulated in fibroblastic foci (p = 0.01, p = 0.08, p = 0.08), the adjacent epithelium (p = 0.001, p = 0.001, p = 0.03), and the hyperplastic type 2 pneumocytes (p = 0.02, p = 0.001, p = 0.08), respectively, compared with control alveoli.

Conclusion: Altered gene expression of important profibrotic mediators in the different cellular lung compartments in patients with UIP likely plays an important role in pathogenesis of the deranged extracellular matrix deposition and subsequent fibrosis in this condition.

Key Words: lung diseases, interstitial • matrix metalloprotease • microdissection • pulmonary fibrosis • tissue inhibitor of matrix metalloprotease-1

Idiopathic pulmonary fibrosis (IPF), a specific form of chronic fibrosing interstitial pneumonia, is associated with the histologic appearance of usual interstitial pneumonia (UIP), which consists of patchy fibrosis, fibroblastic foci, honeycomb change, interstitial smooth muscle proliferation, and hyperplastic type 2 pneumocytes (1, 2). Although the precipitating insult(s) that lead to IPF are unknown, it is postulated that the initial injury relates to abnormal repair of damaged alveolar epithelium manifesting as "abnormal wound repair" (3–6). Alveolar type 2 epithelial cells can undergo hyperplasia and become prominent sources of fibrogenic cytokines (7, 8), providing critical signals to induce fibroblasts to migrate, proliferate, and develop a profibrotic phenotype (6). Without restoration of the alveolar epithelium, there is continuous invasion of fibroblasts and myofibroblasts, and excessive matrix protein deposition, with resulting intraalveolar fibrosis and loss of gas exchange surface area (6).

Fibroblastic foci are independent predictors of lung function and mortality in IPF (9), and are believed to represent early, active areas of aberrant wound repair (1, 10). The fibroblasts and adjacent epithelial cells appear to actively proliferate (11), and can be considered epithelial–mesenchymal trophic units (12). Complex interactions between these cells likely drive the progressive fibrotic process, and understanding this epithelial–mesenchymal cross-talk, and specifically which cytokines and growth factors are involved, is vital to our understanding of the pathology of IPF.

Mediators believed to be important in IPF include transforming growth factor (TGF)-beta1, as well as connective tissue growth factor (CTGF), matrix metalloproteases (MMPs) and their inhibitors (tissue inhibitors of MMPs [TIMPs]), and osteopontin. TGF-beta1 is a key growth factor in the initiation of fibrosis (13), and increased expression of TGF-beta1 mRNA and peptide have been demonstrated in IPF (7, 14). CTGF is directly induced by TGF-beta1 and, in turn, may be essential for the induction of collagen synthesis by TGF-beta (15). An imbalance between the synthesis and degradation of extracellular matrix is central to the progressive fibrosis seen in IPF, and there is evidence that there is a relationship with dysregulation of MMPs and TIMPs (6, 11, 16–18). Osteopontin up-regulates MMP-2 synthesis (19, 20) and promotes fibroblast proliferation and migration (21), whereas osteopontin protein and mRNA expression are up-regulated in bleomycin-induced lung injury in mice and in UIP (18, 20, 22, 23).

The objective of this study was to examine gene expression within specific cell compartments in lung tissue from patients with IPF—namely, the fibroblastic foci, adjacent epithelium, and the hyperplastic type 2 pneumocytes—and compare it with that in alveolar walls from control lungs. Some of the results of this study have been previously reported in the form of an abstract (24).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
The study was approved by the local research ethics committee, and written consent was obtained from all participants. Patients undergoing diagnostic open lung biopsy for suspected IPF on the basis of their clinical and radiologic presentation were invited to participate in the study. Patients with IPF fulfilled the diagnostic criteria of the 2002 American Thoracic Society/European Respiratory Society consensus report (2), and the morphologic diagnosis was based on typical microscopic findings of UIP (1). Histologic assessment confirmed the diagnosis of UIP in 11 patients (10 males; mean age [± SD] = 62 [9.5] yr), and these were included in this study. Tissue samples from 11 patients undergoing resection for lung cancer were included as comparison controls (7 males; mean age [± SD] = 65 [10.4] yr), with the tissue being sampled in areas distant from the tumor.

Handling of Lung Tissue and Laser Capture Microdissection
Details are provided in the online supplement.

Gene Expression in Microdissected Tissue
Extraction of RNA from microdissected tissue was performed as previously described (25) and as described in the online supplement.

Immunohistochemistry
Immunohistochemistry was performed on paraffin-processed tissue to detect TGF-beta1, MMP-7, osteopontin, TIMP-1, and {alpha}-smooth muscle actin ({alpha}-SMA), as described in the online supplement.

Statistical Analysis
Data were analyzed using SPSS version 11.0.0 (SPSS, Inc., Chicago, IL). Reported values are expressed as mean and SEM unless described as otherwise. Normality and variance assumptions were tested for all variables. The gene expression data were nonparametric and were, therefore, logarithmically transformed for analysis. Comparisons between the different tissue samples with respect to gene expression were performed by one-way analysis of variance. Post hoc multiple comparison testing was performed using Bonferroni's test to assess for significant effects. All comparisons were two-tailed, and p values less than 0.05 were considered to be significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Gene Expression of MMPs and TIMP-1 Was Up-regulated within Hyperplastic Type 2 Pneumocytes, Fibroblastic Foci, and Adjacent Epithelium
TIMP-1 and MMP-2 gene expression was significantly increased within the fibroblastic foci compared with the adjacent epithelium (p = 0.03, p = 0.02) and control alveoli (p = 0.001, p = 0.04), respectively (Figure 1). Compared with control alveoli, gene expression of MMP-9 and MMP-7 was up-regulated in the epithelium adjacent to fibroblastic foci (p = 0.001, p = 0.001), and in the hyperplastic type 2 pneumocytes (p = 0.02, p = 0.001), respectively. In addition, gene expression of MMP-9 was up-regulated in the fibroblastic foci (p = 0.01) compared with controls. There was decreased expression of MMP-7 in the fibroblastic foci compared with the adjacent epithelium (p = 0.04).


Figure 1
View larger version (29K):
[in this window]
[in a new window]
  		Figure 1. Relative gene expression of metalloproteases, tissue inhibitor of matrix metalloprotease (TIMP)-1, and osteopontin. Gene expression is relative to beta2-microglobulin; note that the y axis has been logarithmically transformed. MMP = matrix metalloprotease.

 
Gene Expression of Osteopontin Was Up-regulated within Epithelium Adjacent to Fibroblastic Foci
Compared with control alveoli, gene expression of osteopontin was up-regulated in the epithelium adjacent to fibroblastic foci (p = 0.03), whereas enhanced expression showed a trend to significance in the hyperplastic type 2 pneumocytes (p = 0.08) and the fibroblastic foci (p = 0.08; Figure 1).

Gene Expression of TGF-beta1, TGF-beta2, or CTGF Was Not Significantly Up-regulated within Hyperplastic Type 2 Pneumocytes, Fibroblastic Foci, or Adjacent Epithelium
Gene expression of TGF-beta1 and TGF-beta2 was not significantly elevated in any of the selected tissues from patients with IPF, although there was a trend to increased expression in the epithelium adjacent to fibroblastic foci compared with control alveoli (p = 0.09, p = 0.07), respectively (Figure 2). There was also a trend to increased gene expression of CTGF within the fibroblastic foci compared with the adjacent epithelium (p = 0.08).


Figure 2
View larger version (21K):
[in this window]
[in a new window]
  		Figure 2. Relative gene expression of growth factors. Gene expression is relative to beta2-microglobulin. CTGF = connective tissue factor; TGF = transforming growth factor.

 
Gene Expression of {alpha}-SMA in Fibroblastic Foci and Smooth Muscle
To validate the methodology, epithelial and mesenchymal tissues were examined for their expression of {alpha}-SMA and smooth muscle myosin gene expression (Figure 3). The levels of gene expression of {alpha}-SMA within bronchial smooth muscle were significantly increased compared with all epithelial tissues (bronchial epithelium, p = 0.02; hyperplastic type 2 pneumocytes, p < 0.001; epithelium adjacent to fibroblastic foci, p < 0.001), and were also increased with a trend to significance when compared with fibroblastic foci (p = 0.08). The metaplastic smooth muscle within areas of chronic fibrosis also showed increased levels of {alpha}-SMA mRNA compared with bronchial epithelium (p = 0.001), metaplastic epithelium (p = 0.001), and epithelium adjacent to fibroblastic foci (p = 0.003). In addition, {alpha}-SMA expression was up-regulated in fibroblastic foci compared with adjacent epithelium (p = 0.04) and to hyperplastic type 2 pneumocytes (p = 0.01).


Figure 3
View larger version (25K):
[in this window]
[in a new window]
  		Figure 3. Relative gene expression of smooth muscle markers. Gene expression is relative to beta2-microglobulin.

 
Gene Expression of Smooth Muscle Myosin Was Up-regulated only within Bronchial Smooth Muscle
The levels of gene expression of smooth muscle myosin were significantly increased in the bronchial smooth muscle compared with all the other tissues; bronchial epithelium (p = 0.04), hyperplastic type 2 pneumocytes (p = 0.04), epithelium overlying fibroblastic foci (p = 0.04), fibroblastic foci (p = 0.01), and areas of smooth muscle metaplasia (p = 0.02; Figure 3).

Gene Expression of Surfactant Protein C by Alveoli, Hyperplastic Type 2 Pneumocytes, and Epithelium Adjacent to Fibroblastic Foci
Surfactant protein C expression in hyperplastic type 2 pneumocytes was not significantly different from that in alveolar walls (controls), although there was a trend to increased gene expression compared with the epithelium adjacent to fibroblastic foci (p = 0.09). The level of expression in hyperplastic type 2 pneumocytes was significantly higher than that in bronchial epithelium (p = 0.03), fibroblastic foci (0.01), and bronchial smooth muscle (0.01; Figure 4).


Figure 4
View larger version (16K):
[in this window]
[in a new window]
  		Figure 4. Relative gene expression of an epithelial cell marker, surfactant protein C. Gene expression is relative to beta2-microglobulin.

 
No Significant Differences in Gene Expression Were Detected when Mixtures of Tissues Were Examined
When gene expression of TGF-beta1, MMP-7, and TIMP-1 was analyzed from whole tissue sections (multiple tissue types), there was no significant difference between the control or UIP tissues (Figure 5).


Figure 5
View larger version (11K):
[in this window]
[in a new window]
  		Figure 5. Relative gene expression of TGF-beta, MMP-7, and TIMP-1 in multiple tissues. Gene expression is relative to beta2-microglobulin.

 
Protein Expression within the Hyperplastic Type 2 Pneumocytes and Fibroblastic Foci Correlated with Gene Expression
TGF-beta1 protein was present within hyperplastic type 2 pneumocytes and in the epithelium overlying the fibroblastic foci, but not in the fibroblastic foci (Figures 6A and 6B). Similarly, MMP-7, osteopontin, and TIMP-1 protein were present within hyperplastic type 2 pneumocytes, the epithelium overlying the fibroblastic foci, and within myofibroblasts contained in the fibroblastic foci (Figures 6C–6G). Staining for {alpha}-SMA protein was strongly positive within the fibroblastic foci (Figure 6H), as well as in airway and vascular smooth muscle and in metaplastic smooth muscle present in areas of fibrosis. Normal alveolar walls were negative for MMP-7, but showed scattered, weak staining for TGF-beta1, osteopontin, and TIMP-1, as did airway epithelium.


Figure 6
View larger version (98K):
[in this window]
[in a new window]
  		Figure 6. Immunohistochemistry for TGF-beta1, MMP-7, osteopontin, TIMP-1, and {alpha}-smooth muscle actin ({alpha}-SMA). (A) TGF-beta1 in epithelium overlying fibroblastic focus; cells within fibroblastic focus are negative. (B) TGF-beta1 in hyperplastic type 2 pneumocytes. (C) MMP-7 in fibroblastic focus as well as overlying epithelium. (D) MMP-7 in hyperplastic type 2 pneumocytes. (E) Osteopontin in fibroblastic focus as well as overlying epithelium. (F) Osteopontin in hyperplastic type 2 pneumocytes. (G) TIMP-1 in fibroblastic focus and overlying epithelium. (H) {alpha}-SMA within fibroblastic focus but overlying epithelium is unstained. Original magnification of all photomicrographs, 400x. Solid arrows point to epithelium overlying fibroblastic foci in A, C, E, G, and H, and to hyperplastic type 2 pneumocytes in B, D, and F; open arrows point to the centers of fibroblastic foci.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is the first study to report on cell-specific gene expression in UIP in a quantitative manner. We demonstrated significant up-regulation of gene expression within fibroblastic foci of TIMP-1, MMP-2, and MMP-9, with a trend to significance of MMP-7 and osteopontin compared with control alveoli (Figure 1). The epithelium adjacent to the fibroblastic foci and the hyperplastic type 2 pneumocytes also showed significant up-regulation of MMP-9 and MMP-7 compared with control alveoli; osteopontin gene expression was significantly up-regulated in the epithelium adjacent to the fibroblastic foci compared with control alveoli. Although MMP-7 expression was significantly increased in the fibroblastic foci compared with control alveoli, it was significantly less than within the epithelium overlying the fibroblastic foci. However, there was increased TIMP-1 and MMP-2 mRNA in the fibroblastic foci compared with the overlying epithelium (Figure 1). We were unable to detect significant upregulation of TGF-beta1, TGF-beta2, or CTGF gene expression in individual cell types or whole UIP tissue (Figure 2).

To further validate our methodologic approach, we measured the expression of genes specific to certain cell types—namely, {alpha}-SMA and smooth muscle myosin (both specific for smooth muscle) and surfactant protein C (specific for type 2 pneumocytes)—and confirmed these genes to be present in the relevant cell types (Figures 3 and 4). We also performed immunohistochemistry to detect immunoreactive protein for TGF-beta1, MMP-7, osteopontin, TIMP-1, and {alpha}-SMA. These results correlated with the gene expression data (Figure 6), thus providing additional validation of those data.

MMPs are bound to cell surfaces, and because their substrate specificity and activity is dictated by the cell expressing them (26–28), it is relevant to distinguish their expression by different cell types. Our observed dysregulation of MMP and TIMP-1 gene expression is in keeping with previous studies in experimental fibrosis (29, 30) and UIP (11, 31), in which a nondegrading extracellular matrix environment is the dominant feature. Despite this, lung biopsy homogenates in UIP have failed to demonstrate up-regulated TIMP gene expression (18, 22). This failure may be due to the signal from the fibroblastic foci being masked by those from the rest of the lung, and, indeed, we found no significant difference in TIMP-1 gene expression when we analyzed tissue consisting of multiple cell populations (Figure 5). In addition to its inhibitory effects on MMPs, TIMP-1 inhibits fibroblast and myofibroblast apoptosis (32–36), and also directly promotes fibroblast growth (37–40). The observed up-regulation of TIMP-1 gene expression and presence of the protein in myofibroblasts in fibroblastic foci suggests that TIMP-1 mediates persistence of myofibroblastic differentiation in these foci, even when the overlying epithelium has been lost.

Previous studies demonstrated up-regulated expression of MMP-2 and MMP-9 mRNA in UIP lung homogenates (18, 22), and MMP-2 and MMP-9 protein presence in myofibroblasts and areas of denuded alveolar basement membrane in UIP (11, 41). Aside from the effect of MMPs in enhancing extracellular matrix turnover and promoting tissue remodeling, the expression of these gelatinases in myofibroblasts may facilitate their migration through epithelial basement membrane and aid in invasion of the alveoli (11, 31). MMP-2 and MMP-9 have been shown to play a role in smooth muscle cell migration after injury (42, 43), with MMP-9 also affecting smooth muscle proliferation (42) and collagen assembly (44). MMP-2 can activate MMP-9 (45), and both MMP-2 and MMP-9 can activate TGF-beta (46, 47). Paradoxically, both MMP-2 and MMP-9 can also up-regulate expression of TIMP-1, thereby preventing apoptosis of myofibroblasts. In addition, increased expression of MMP-2 within the fibroblastic foci may stimulate proliferation of fibroblasts within these same foci (48). MMPs also have effects on the release of other profibrotic factors, such as insulin-like growth factor and tumor necrosis factor-{alpha} (47, 49). Although most MMPs promote epithelial repair (50), MMP-9 is unique in that it inhibits cell replication in the migrating epithelial sheet (51), and patients with rapid progression may reflect excess MMP-9 activity (52).

Our observation that MMP-7 gene expression is up-regulated in hyperplastic type 2 pneumocytes and epithelium adjacent to fibroblastic foci is in keeping with other studies, which have detected MMP-7 protein expression within alveolar pneumocytes, the interstitium, and fibroblastic foci in UIP (17, 18). MMP-7 increases the secretion and activation of pro–MMP-2 and pro–MMP-9 (53). Increased local procoagulant activity, attributed to the alveolar epithelial cells, has been observed in IPF (6), and MMP-7 and MMP-9 are able to rapidly cleave tissue factor pathway inhibitor, an inhibitor of tissue factor–induced coagulation (54). MMP-7 may also be involved in the release of the profibrotic cytokine tumor necrosis factor-{alpha} (55, 56). After epithelial injury, MMP-7 causes shedding of epithelial cadherin (E-cadherin), an important mediator of epithelial cell adhesion, and, in IPF, there is persistent shedding of E-cadherin with consequent failure of epithelial repair (57). There is also evidence of aberrant activation of the Wnt pathway in hyperplastic type 2 pneumocytes and within the fibroblastic foci in UIP (58), which could provide autocrine survival signals to the fibroblasts/myofibroblasts within the fibroblastic foci (59–61). MMP-7 is itself induced by the Wnt/beta-catenin signaling pathway (62), and may, thereby, initiate a positive feedback loop for its own gene expression in injured epithelium (60).

Microarray studies have shown significant interaction between osteopontin and MMP-7 (63), and, in UIP, osteopontin protein has been demonstrated in alveolar epithelium and hyperplastic type 2 pneumocytes, but not within fibroblastic foci (23). Although osteopontin has previously been shown to be a TGF-beta1 response gene (64), it may also function upstream of TGF-beta1 by regulating its activation (22, 23, 65), and it can also activate MMP-2 (66). When treated with bleomycin, osteopontin-deficient mice express less MMP-2 and active TGF-beta1 (23), and osteopontin may play an important amplifying or initiating role in up-regulating MMP-7 in epithelial cells and TIMP-1 in fibroblasts (63). In our study, osteopontin gene expression was up-regulated within fibroblastic foci, which correlated with protein expression (Figure 6E).

Our results support the hypothesis that myofibroblasts within the fibroblastic foci are able to penetrate the overlying epithelial basement membrane through their ability to produce MMP-2 and MMP-9. Attempts to re-epithelialize the fibroblastic foci may be hindered by high levels of MMP-7 and MMP-9 secreted by the epithelial cells themselves and by the myofibroblasts within the fibroblastic foci, which results in persistent epithelial disruption. The "injured" epithelium chronically produces MMP-7, MMP-9, and osteopontin, and, together with TIMP-1 production by the fibroblastic foci, results in a persistent imbalance in collagen synthesis and degradation, with progressive enlargement of the fibroblastic foci.

Despite TGF-beta1 being strongly associated with UIP (67, 68), and TGF-beta mRNA and protein having been found at increased levels within the sites of extracellular matrix accumulation (69), we did not find a significant up-regulation of TGF-beta1 or TGF-beta2 within the fibroblastic foci in our study. TGF-beta1 protein was demonstrated to be present within hyperplastic type 2 pneumocytes and the epithelium overlying the fibroblastic foci, but not within the fibroblastic foci itself (Figures 6A and 6B), which supports the gene expression findings. Similarly, a previous study showed that immunoreactive TGF-beta1 was present in alveolar macrophages and epithelial cells of patients with IPF, but was absent in fibroblasts in the subepithelial regions of honeycomb cysts (70). In addition, a study of UIP using in situ hybridization demonstrated that TGF-beta1 mRNA was present in macrophages adjacent to fibroblastic foci, but that the foci themselves showed no signal for TGF-beta1 mRNA (14). Global gene profiling of lung treated with bleomycin in experimental fibrosis has also failed to detect a significant increase in TGF-beta mRNA, despite marked up-regulation of numerous TGF-beta1–inducible genes (22). The lower respiratory tract usually contains high levels of latent TGF-beta1 (71), although macrophages, rather than epithelium, appear to be the primary source of TGF-beta1 (72). Although structural cells in UIP may not be the source of increased TGF-beta1, they may activate matrix-associated TGF-beta1 protein through their production of MMP-2, MMP-9, and osteopontin, resulting in persistent expression of genes relating to tissue fibrosis (47, 73). Thus, our findings do not exclude a role for TGF-beta1 in initiating myofibroblast development within fibroblastic foci, and these myofibroblasts may be more responsive to growth factors such as TGF-beta1 and CTGF (74).

We did not observe a significant difference in CTGF gene expression between fibroblastic foci and control alveoli, although there was a trend toward significantly increased mRNA within the fibroblastic foci compared with the adjacent epithelium. Although a previous study has shown increased expression of CTGF mRNA and protein in alveolar type 2 cells and interstitial fibroblasts in UIP (75), we have previously found only transient fibrosis on adenovirus vector transfer of CTGF to the rodent lung (76). Our finding of significant up-regulation of TIMP-1, but not CTGF, within the fibroblastic foci suggests decreased degradation of collagen, rather than enhanced synthesis, as the mechanism for the fibrosis seen in UIP.

As expected, we found that the smooth muscle cell genes, {alpha}-SMA and smooth muscle myosin, were present at a high level within bronchial smooth muscle, and {alpha}-SMA also showed significant up-regulation in fibroblastic foci and the areas of metaplastic smooth muscle (Figure 3). We did not find up-regulation of {alpha}-SMA gene or protein expression in the epithelium adjacent to the fibroblastic foci, despite a recent study suggesting epithelial–mesenchymal transition in IPF (77). Similarly, surfactant protein C, a gene specific to type 2 pneumocytes, was present at a high level in the control alveoli and the hyperplastic type 2 pneumocytes only (Figure 4). Mutations in the surfactant protein C gene have been linked to familial forms of pulmonary fibrosis (78), and it has recently been shown that surfactant protein C–deficient mice had increased fibrosis with an associated delay in fibrosis resolution in a bleomycin-induced pulmonary fibrosis model (79). Interestingly, in our study, there was a trend toward a significant reduction in surfactant protein C gene expression in the epithelium adjacent to the fibroblastic foci compared with the hyperplastic type 2 pneumocytes. This may reflect a more severe injury in these cells.

When mRNA was extracted from multiple cell types present in a total tissue section, the differences in gene regulation seen between the UIP and control samples were lost (Figure 5), emphasizing the unique information gained from this methodology. Laser capture microdissection with quantitative RT-PCR has the advantages over in situ hybridization of allowing the examination of several different genes simultaneously and providing a more sensitive, quantitative measurement. Alveoli distant from tumor-free areas of lung resected for lung cancer were chosen as the control in this study, as UIP affects the distal pulmonary acinus and there is evidence that alveolar pneumocytes are the cell type initiating the disease (80). Although gene expression studies cannot provide information on post-translational modifications of proteins, this study demonstrates the validity and usefulness of being able to examine gene expression in specific cell populations in the lung in UIP.

Useful as animal models of pulmonary fibrosis are, they show significant differences in pathogenesis and outcome when compared with IPF (81), and, therefore, studies on human subjects with IPF are important. Gene microarray studies in IPF have yielded valuable information (17, 18), but are limited by their inability to determine which cells are responsible for specific gene overexpression. Changes in gene expression may merely reflect changes in cellular composition, and signals from relatively small areas, such as fibroblastic foci, may be masked by quantitatively stronger signals from other tissues. Because many genes are multifunctional, it is important to assess their expression in the correct tissue context. By examining gene expression within fibroblastic foci, the adjacent epithelium, and in areas of hyperplastic type 2 pneumocytes, we believe that we were able to examine early events in this chronic but temporally heterogeneous disease. The similarities in the gene expression profile between the hyperplastic type 2 pneumocytes and the epithelium overlying the fibroblastic foci suggest that type 2 cells may represent injured epithelium, which subsequently initiates the fibroblastic response, rather than constituting a nonspecific reactive process. Type 2 pneumocytes have direct contact with underlying fibroblasts (82), and it has been suggested that an underlying defect in the type 2 pneumocyte may be responsible for initiation of UIP (6, 83, 84). Our findings of up-regulated gene expression of MMP-7, -9, and osteopontin within the epithelium adjacent to fibroblastic foci may represent a mechanism of persistence, or even initiation, of these foci. Osteopontin and MMP-7 may be key molecules in this regard, as they are upregulated in both the epithelium adjacent to the fibroblastic foci and in the hyperplastic type 2 epithelium.

Our findings of significantly up-regulated TIMP-1 expression within the fibroblastic foci suggest that decreased collagen breakdown, due to the presence of a nondegrading environment (11), is a more important pathogenetic mechanism than excessive collagen synthesis in the evolution of this disease. As such, new therapies may be better targeted at reversing the inhibition of collagen degradation rather than preventing the synthesis of new collagen.


   Acknowledgments
 
The authors thank Carol Lavery, Mary Jo Smith, and Carol Gwozd for excellent technical help.


   FOOTNOTES
 
Supported by an operating grant from the Canadian Institutes for Health Research. M.M.K. and R.L. are recipients of Canadian Institutes for Health Research Fellowships.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200510-1648OC on May 25, 2006

Conflict of Interest Statement: M.M.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.L. received $80,000 as a grant-in-aid from GlaxoSmithKline in 2004. He also received $1,500 in 2005 for serving on an advisory board for AstraZeneca. In addition, he has participated as a speaker in scientific and educational meetings organized and financed by GlaxoSmithKline, AstraZeneca, Altana, Pfizer, Boehringer-Ingelheim, and Sanofi-Aventis throughout 2004 and 2005. S.E.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. E.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. G.E.M.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. G.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form October 21, 2005; accepted in final form May 25, 2006


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Katzenstein AL, Myers JL. Idiopathic pulmonary fibrosis: clinical relevance of pathologic classification. Am J Respir Crit Care Med 1998;157: 1301–1315.[Free Full Text]
  2. American Thoracic Society/European Respiratory Society. American Thoracic Society/European Respiratory Society International Multidisciplinary Consensus Classification of the Idiopathic Interstitial Pneumonias. Am J Respir Crit Care Med 2002;165:277–304.[Free Full Text]
  3. Myers JL, Katzenstein AL. Ultrastructural evidence of alveolar epithelial injury in idiopathic bronchiolitis obliterans-organizing pneumonia. Am J Pathol 1988;132:102–109.[Abstract]
  4. Fang KC. Mesenchymal regulation of alveolar repair in pulmonary fibrosis. Am J Respir Cell Mol Biol 2000;23:142–145.[Free Full Text]
  5. Morishima Y, Nomura A, Uchida Y, Noguchi Y, Sakamoto T, Ishii Y, Goto Y, Masuyama K, Zhang MJ, Hirano K, et al. Triggering the induction of myofibroblast and fibrogenesis by airway epithelial shedding. Am J Respir Cell Mol Biol 2001;24:1–11.[Abstract/Free Full Text]
  6. Selman M, King TE, Pardo A. Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann Intern Med 2001;134:136–151.[Abstract/Free Full Text]
  7. Khalil N, O'Connor RN, Flanders KC, Unruh H. TGF-beta 1, but not TGF-beta 2 or TGF-beta 3, is differentially present in epithelial cells of advanced pulmonary fibrosis: an immunohistochemical study. Am J Respir Cell Mol Biol 1996;14:131–138.[Abstract]
  8. Fehrenbach H. Alveolar epithelial type II cell: defender of the alveolus revisited. Respir Res 2001;2:33–46.[Medline]
  9. King TE Jr, Schwarz MI, Brown K, Tooze JA, Colby TV, Waldron JA Jr, Flint A, Thurlbeck W, Cherniack RM. Idiopathic pulmonary fibrosis: relationship between histopathologic features and mortality. Am J Respir Crit Care Med 2001;164:1025–1032.[Abstract/Free Full Text]
  10. Kuhn C, McDonald JA. The roles of the myofibroblast in idiopathic pulmonary fibrosis: ultrastructural and immunohistochemical features of sites of active extracellular matrix synthesis. Am J Pathol 1991;138: 1257–1265.[Abstract]
  11. Selman M, Ruiz V, Cabrera S, Segura L, Ramirez R, Barrios R, Pardo A. TIMP-1, -2, -3, and -4 in idiopathic pulmonary fibrosis: a prevailing nondegradative lung microenvironment? Am J Physiol Lung Cell Mol Physiol 2000;279:L562–L574.[Abstract/Free Full Text]
  12. Evans MJ, Van Winkle LS, Fanucchi MV, Plopper CG. The attenuated fibroblast sheath of the respiratory tract epithelial–mesenchymal trophic unit. Am J Respir Cell Mol Biol 1999;21:655–657.[Free Full Text]
  13. Sime PJ, Xing Z, Graham FL, Csaky KG, Gauldie J. Adenovector-mediated gene transfer of active transforming growth factor-beta1 induces prolonged severe fibrosis in rat lung. J Clin Invest 1997;100:768–776.[Medline]
  14. Broekelmann TJ, Limper AH, Colby TV, McDonald JA. Transforming growth factor beta1 is present at sites of extracellular matrix gene expression in human pulmonary fibrosis. Proc Natl Acad Sci USA 1991;88: 6642–6646.[Abstract/Free Full Text]
  15. Duncan MR, Frazier KS, Abramson S, Williams S, Klapper H, Huang X, Grotendorst GR. Connective tissue growth factor mediates transforming growth factor beta-induced collagen synthesis: down-regulation by cAMP. FASEB J 1999;13:1774–1786.[Abstract/Free Full Text]
  16. Lemjabbar H, Gosset P, Lechapt-Zalcman E, Franco-Montoya ML, Wallaert B, Harf A, Lafuma C. Overexpression of alveolar macrophage gelatinase B (MMP-9) in patients with idiopathic pulmonary fibrosis: effects of steroid and immunosuppressive treatment. Am J Respir Cell Mol Biol 1999;20:903–913.[Abstract/Free Full Text]
  17. Cosgrove GP, Schwarz MI, Geraci MW, Brown KK, Worthen GS. Overexpression of matrix metalloproteinase-7 in pulmonary fibrosis. Chest 2002;121:25S–26S.
  18. Zuo F, Kaminski N, Eugui E, Allard J, Yakhini Z, Ben Dor A, Lollini L, Morris D, Kim Y, DeLustro B, et al. Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans. Proc Natl Acad Sci USA 2002;99:6292–6297.[Abstract/Free Full Text]
  19. Seipelt RG, Backer CL, Mavroudis C, Stellmach V, Cornwell M, Seipelt IM, Schoendube FA, Crawford SE. Local delivery of osteopontin attenuates vascular remodeling by altering matrix metalloproteinase-2 in a rabbit model of aortic injury. J Thorac Cardiovasc Surg 2005;130: 355–362.[Abstract/Free Full Text]
  20. Takahashi F, Takahashi K, Okazaki T, Maeda K, Ienaga H, Maeda M, Kon S, Uede T, Fukuchi Y. Role of osteopontin in the pathogenesis of bleomycin-induced pulmonary fibrosis. Am J Respir Cell Mol Biol 2001;24:264–271.[Abstract/Free Full Text]
  21. Philip S, Bulbule A, Kundu GC. Matrix metalloproteinase-2: mechanism and regulation of NF-kappaB–mediated activation and its role in cell motility and ECM-invasion. Glycoconj J 2004;21:429–441.[CrossRef][Medline]
  22. Kaminski N, Allard JD, Pittet JF, Zuo F, Griffiths MJ, Morris D, Huang X, Sheppard D, Heller RA. Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and fibrosis. Proc Natl Acad Sci USA 2000;97:1778–1783.[Abstract/Free Full Text]
  23. Berman JS, Serlin D, Li X, Whitley G, Hayes J, Rishikof DC, Ricupero DA, Liaw L, Goetschkes M, O'Regan AW. Altered bleomycin-induced lung fibrosis in osteopontin-deficient mice. Am J Physiol Lung Cell Mol Physiol 2004;286:L1311–L1318.[Abstract/Free Full Text]
  24. Kelly MM, Leigh R, Gilpin SE, Cheng EV, Martin GEM, Radford K, Cox PG, Gauldie J. Cell specific gene expression in IPF lung. Am J Respir Crit Care Med 2005;2:A637.
  25. Kelly MM, Leigh R, Bonniaud P, Ellis R, Wattie J, Smith MJ, Martin G, Panju M, Inman MD, Gauldie J. Epithelial expression of profibrotic mediators in a model of allergen-induced airway remodeling. Am J Respir Cell Mol Biol 2005;32:99–107.[Abstract/Free Full Text]
  26. Brooks PC, Stromblad S, Sanders LC, von Schalscha TL, Aimes RT, Stetler-Stevenson WG, Quigley JP, Cheresh DA. Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin {alpha}vbeta3. Cell 1996;85:683–693.[CrossRef][Medline]
  27. Yu Q, Stamenkovic I. Localization of matrix metalloproteinase 9 to the cell surface provides a mechanism for CD44-mediated tumor invasion. Genes Dev 1999;13:35–48.[Abstract/Free Full Text]
  28. Yu WH, Woessner JF Jr, McNeish JD, Stamenkovic I. CD44 anchors the assembly of matrilysin/MMP-7 with heparin-binding epidermal growth factor precursor and ErbB4 and regulates female reproductive organ remodeling. Genes Dev 2002;16:307–323.[Abstract/Free Full Text]
  29. Swiderski RE, Dencoff JE, Floerchinger CS, Shapiro SD, Hunninghake GW. Differential expression of extracellular matrix remodeling genes in a murine model of bleomycin-induced pulmonary fibrosis. Am J Pathol 1998;152:821–828.[Abstract]
  30. Madtes DK, Elston AL, Kaback LA, Clark JG. Selective induction of tissue inhibitor of metalloproteinase-1 in bleomycin-induced pulmonary fibrosis. Am J Respir Cell Mol Biol 2001;24:599–607.[Abstract/Free Full Text]
  31. Hayashi T, Stetler-Stevenson WG, Fleming MV, Fishback N, Koss MN, Liotta LA, Ferrans VJ, Travis WD. Immunohistochemical study of metalloproteinases and their tissue inhibitors in the lungs of patients with diffuse alveolar damage and idiopathic pulmonary fibrosis. Am J Pathol 1996;149:1241–1256.[Abstract]
  32. Valente P, Fassina G, Melchiori A, Masiello L, Cilli M, Vacca A, Onisto M, Santi L, Stetler-Stevenson WG, Albini A. TIMP-2 over-expression reduces invasion and angiogenesis and protects B16F10 melanoma cells from apoptosis. Int J Cancer 1998;75:246–253.[CrossRef][Medline]
  33. Guedez L, Stetler-Stevenson WG, Wolff L, Wang J, Fukushima P, Mansoor A, Stetler-Stevenson M. In vitro suppression of programmed cell death of b cells by tissue inhibitor of metalloproteinases-1. J Clin Invest 1998;102:2002–2010.[Medline]
  34. Alexander CM, Howard EW, Bissell MJ, Werb Z. Rescue of mammary epithelial cell apoptosis and entactin degradation by a tissue inhibitor of metalloproteinases-1 transgene. J Cell Biol 1996;135:1669–1677.[Abstract/Free Full Text]
  35. Baker AH, Zaltsman AB, George SJ, Newby AC. Divergent effects of tissue inhibitor of metalloproteinase-1, -2, or -3 overexpression on rat vascular smooth muscle cell invasion, proliferation, and death in vitro: TIMP-3 promotes apoptosis. J Clin Invest 1998;101:1478–1487.[Medline]
  36. Murphy FR, Issa R, Zhou X, Ratnarajah S, Nagase H, Arthur MJ, Benyon C, Iredale JP. Inhibition of apoptosis of activated hepatic stellate cells by TIMP-1 is mediated via effects on MMP inhibition: implications for reversibility of liver fibrosis. J Biol Chem 2002;277:11069–11076.[Abstract/Free Full Text]
  37. Hayakawa T, Yamashita K, Tanzawa K, Uchijima E, Iwata K. Growth-promoting activity of tissue inhibitor of metalloproteinases-1 (TIMP-1) for a wide range of cells: a possible new growth factor in serum. FEBS Lett 1992;298:29–32.[CrossRef][Medline]
  38. Stetler-Stevenson WG, Bersch N, Golde DW. Tissue inhibitor of metalloproteinase-2 (TIMP-2) has erythroid-potentiating activity. FEBS Lett 1992;296:231–234.[CrossRef][Medline]
  39. Hayakawa T, Yamashita K, Ohuchi E, Shinagawa A. Cell growth–promoting activity of tissue inhibitor of metalloproteinases-2 (TIMP-2). J Cell Sci 1994;107:2373–2379.[Abstract]
  40. Chesler L, Golde DW, Bersch N, Johnson MD. Metalloproteinase inhibition and erythroid potentiation are independent activities of tissue inhibitor of metalloproteinases-1. Blood 1995;86:4506–4515.[Abstract/Free Full Text]
  41. Fukuda Y, Ishizaki M, Kudoh S, Kitaichi M, Yamanaka N. Localization of matrix metalloproteinases-1, -2, and -9 and tissue inhibitor of metalloproteinase-2 in interstitial lung diseases. Lab Invest 1998;78: 687–698.[Medline]
  42. Cho A, Reidy MA. Matrix Metalloproteinase-9 is necessary for the regulation of smooth muscle cell replication and migration after arterial injury. Circ Res 2002;91:845–851.[Abstract/Free Full Text]
  43. Kuzuya M, Kanda S, Sasaki T, Tamaya-Mori N, Cheng XW, Itoh T, Itohara S, Iguchi A. Deficiency of gelatinase A suppresses smooth muscle cell invasion and development of experimental intimal hyperplasia. Circulation 2003;108:1375–1381.[Abstract/Free Full Text]
  44. Johnson C, Galis ZS. Matrix metalloproteinase-2 and -9 differentially regulate smooth muscle cell migration and cell-mediated collagen organization. Arterioscler Thromb Vasc Biol 2004;24:54–60.[Abstract/Free Full Text]
  45. Ogata Y, Enghild JJ, Nagase H. Matrix metalloproteinase 3 (stromelysin) activates the precursor for the human matrix metalloproteinase 9. J Biol Chem 1992;267:3581–3584.[Abstract/Free Full Text]
  46. Wang L, Clutter S, Benincosa J, Fortney J, Gibson LF. Activation of transforming growth factor-beta1/p38/Smad3 signaling in stromal sells requires reactive oxygen species-mediated MMP-2 activity during bone marrow damage. Stem Cells 2005;23:1122–1134.[Abstract/Free Full Text]
  47. Yu Q, Stamenkovic I. Cell surface–localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis. Genes Dev 2000;14:163–176.[Abstract/Free Full Text]
  48. Xu J, Benyon RC, Leir SH, Zhang S, Holgate ST, Lackie PM. Matrix metalloproteinase-2 from bronchial epithelial cells induces the proliferation of subepithelial fibroblasts. Clin Exp Allergy 2002;32:881–888.[CrossRef][Medline]
  49. Blobel CP. Remarkable roles of proteolysis on and beyond the cell surface. Curr Opin Cell Biol 2000;12:606–612.[CrossRef][Medline]
  50. Vu TH, Werb Z. Matrix metalloproteinases: effectors of development and normal physiology. Genes Dev 2000;14:2123–2133.[Free Full Text]
  51. Mohan R, Chintala SK, Jung JC, Villar WVL, McCabe F, Russo LA, Lee Y, McCarthy BE, Wollenberg KR, Jester JV, et al. Matrix metalloproteinase gelatinase B (MMP-9) coordinates and effects epithelial regeneration. J Biol Chem 2002;277:2065–2072.[Abstract/Free Full Text]
  52. Atkinson JJ, Senior RM. Matrix metalloproteinase-9 in lung remodeling. Am J Respir Cell Mol Biol 2003;28:12–24.[Abstract/Free Full Text]
  53. Wang, F, Reierstad S, Fishman DA. Matrilysin over-expression in MCF-7 cells enhances cellular invasiveness and pro-gelatinase activation. Cancer Lett 2006;236:292–301.[CrossRef][Medline]
  54. Belaaouaj AA, Li A, Wun TC, Welgus HG, Shapiro SD. Matrix metalloproteinases cleave tissue factor pathway inhibitor: effects on coagulation. J Biol Chem 2000;275:27123–27128.[Abstract/Free Full Text]
  55. Allen JT, Spiteri MA. Growth factors in idiopathic pulmonary fibrosis: relative roles. Respir Res 2002;3:13.[Medline]
  56. Haro H, Crawford HC, Fingleton B, Shinomiya K, Spengler DM, Matrisian LM. Matrix metalloproteinase-7–dependent release of tumor necrosis factor-{alpha} in a model of herniated disc resorption. J Clin Invest 2000;105:143–150.[Medline]
  57. McGuire JK, Li Q, Parks WC. Matrilysin (matrix metalloproteinase-7) mediates E-cadherin ectodomain shedding in injured lung epithelium. Am J Pathol 2003;162:1831–1843.[Abstract/Free Full Text]
  58. Chilosi M, Poletti V, Zamo A, Lestani M, Montagna L, Piccoli P, Pedron S, Bertaso M, Scarpa A, Murer B, et al. Aberrant Wnt/beta-catenin pathway activation in idiopathic pulmonary fibrosis. Am J Pathol 2003;162:1495–1502.[Abstract/Free Full Text]
  59. Ueda Y, Hijikata M, Takagi S, Takada R, Takada S, Chiba T, Shimotohno K. Wnt/beta-catenin signaling suppresses apoptosis in low serum medium and induces morphologic change in rodent fibroblasts. Int J Cancer 2002;99:681–688.[CrossRef][Medline]
  60. Cheon S, Poon R, Yu C, Khoury M, Shenker R, Fish J, Alman BA. Prolonged beta-catenin stabilization and tcf-dependent transcriptional activation in hyperplastic cutaneous wounds. Lab Invest 2005;85:416–425.[CrossRef][Medline]
  61. Douglas IS, Diaz del Valle F, Winn RA, Voelkel NF. beta-Catenin in the fibroproliferative response to acute lung injury. Am J Respir Cell Mol Biol 2006;34:274–285.[Abstract/Free Full Text]
  62. Crawford HC, Fingleton BM, Rudolph-Owen LA, Goss KJ, Rubinfeld B, Polakis P, Matrisian LM. The metalloproteinase matrilysin is a target of beta-catenin transactivation in intestinal tumors. Oncogene 1999;18:2883–2891.[CrossRef][Medline]
  63. Pardo A, Gibson K, Cisneros J, Richards TJ, Yang Y, Becerril C, Yousem S, Herrera I, Ruiz V, Selman M, et al. Up-regulation and profibrotic role of osteopontin in human idiopathic pulmonary fibrosis. PLoS Med 2005;2:e251.[CrossRef][Medline]
  64. Fagenholz PJ, Warren SM, Greenwald JA, Bouletreau PJ, Spector JA, Crisera FE, Longaker MT. Osteoblast gene expression is differentially regulated by TGF-beta isoforms. J Craniofac Surg 2001;12:183–190.[CrossRef][Medline]
  65. Denhardt DT, Noda M, O'Regan AW, Pavlin D, Berman JS. Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival. J Clin Invest 2001;107:1055–1061.[Medline]
  66. Philip S, Bulbule A, Kundu GC. Osteopontin stimulates tumor growth and activation of promatrix metalloproteinase-2 through nuclear factor-{kappa}B–mediated induction of membrane type 1 matrix metalloproteinase in murine melanoma cells. J Biol Chem 2001;276:44926–44935.[Abstract/Free Full Text]
  67. Khalil N, O'Connor RN, Unruh HW, Warren PW, Flanders KC, Kemp A, Bereznay OH, Greenberg AH. Increased production and immunohistochemical localization of transforming growth factor-beta in idiopathic pulmonary fibrosis. Am J Respir Cell Mol Biol 1991;5:155–162.[Medline]
  68. Coker RK, Laurent GJ, Jeffery PK, du Bois RM, Black CM, McAnulty RJ. Localisation of transforming growth factor beta1 and beta3 mRNA transcripts in normal and fibrotic human lung. Thorax 2001;56:549–556.[Abstract/Free Full Text]
  69. Lasky JA, Brody AR. Interstitial fibrosis and growth factors. Environ Health Perspect 2000;108:751–762.[CrossRef][Medline]
  70. Khalil N, Parekh TV, O'Connor R, Antman N, Kepron W, Yehaulaeshet T, Xu YD, Gold LI. Regulation of the effects of TGF-beta 1 by activation of latent TGF-beta 1 and differential expression of TGF-beta receptors (T beta R-I and T beta R-II) in idiopathic pulmonary fibrosis. Thorax 2001; 56:907–915.[Abstract/Free Full Text]
  71. Yamauchi K, Martinet Y, Basset P, Fells GA, Crystal RG. High levels of transforming growth factor-beta are present in the epithelial lining fluid of the normal human lower respiratory tract. Am Rev Respir Dis 1988;137:1360–1363.[Medline]
  72. Khalil N, Bereznay O, Sporn M, Greenberg AH. Macrophage production of transforming growth factor beta and fibroblast collagen synthesis in chronic pulmonary inflammation. J Exp Med 1989;170:727–737.[Abstract/Free Full Text]
  73. Kunugi S, Fukuda Y, Ishizaki M, Yamanaka N. Role of MMP-2 in alveolar epithelial cell repair after bleomycin administration in rabbits. Lab Invest 2001;81:1309–1318.[Medline]
  74. Hagood JS, Prabhakaran P, Kumbla P, Salazar L, MacEwen MW, Barker TH, Ortiz LA, Schoeb T, Siegal GP, Alexander CB, et al. Loss of fibroblast Thy-1 expression correlates with lung fibrogenesis. Am J Pathol 2005;167:365–379.[Abstract/Free Full Text]
  75. Pan LH, Yamauchi K, Uzuki M, Nakanishi T, Takigawa M, Inoue H, Sawai T. Type II alveolar epithelial cells and interstitial fibroblasts express connective tissue growth factor in IPF. Eur Respir J 2001; 17:1220–1227.[Abstract/Free Full Text]
  76. Bonniaud P, Margetts PJ, Kolb M, Haberberger T, Kelly M, Robertson J, Gauldie J. Adenoviral gene transfer of connective tissue growth factor in the lung induces transient fibrosis. Am J Respir Crit Care Med 2003;168:770–778.[Abstract/Free Full Text]
  77. Willis BC, Liebler JM, Luby-Phelps K, Nicholson AG, Crandall ED, du Bois RM, Borok Z. Induction of epithelial–mesenchymal transition in alveolar epithelial cells by transforming growth factor-beta1: potential role in idiopathic pulmonary fibrosis. Am J Pathol 2005;166:1321–1332.[Abstract/Free Full Text]
  78. Grutters JC, du Bois RM. Genetics of fibrosing lung diseases. Eur Respir J 2005;25:915–927.[Abstract/Free Full Text]
  79. Lawson WE, Polosukhin VV, Stathopoulos GT, Zoia O, Han W, Lane KB, Li B, Donnelly EF, Holburn GE, Lewis KG, et al. Increased and prolonged pulmonary fibrosis in surfactant protein C–deficient mice following intratracheal bleomycin. Am J Pathol 2005;167:1267–1277.[Abstract/Free Full Text]
  80. Myers JL, Selman M. Respiratory epithelium in usual interstitial pneumonia/idiopathic pulmonary fibrosis: spark or destructive flame? Am J Respir Crit Care Med 2004;169:3–5.[Free Full Text]
  81. Borzone G, Moreno R, Urrea R, Meneses M, Oyarzun M, Lisboa C. Bleomycin-induced chronic lung damage does not resemble human idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2001;163: 1648–1653.[Abstract/Free Full Text]
  82. Sirianni FE, Chu FS, Walker DC. Human alveolar wall fibroblasts directly link epithelial type 2 cells to capillary endothelium. Am J Respir Crit Care Med 2003;168:1532–1537.[Abstract/Free Full Text]
  83. Williams K, Malarkey D, Cohn L, Patrick D, Dye J, Toews G. Identification of spontaneous feline idiopathic pulmonary fibrosis: morphology and ultrastructural evidence for a type II pneumocyte defect. Chest 2004;125:2278–2288.[Medline]
  84. Kasper M, Haroske G. Alterations in the alveolar epithelium after injury leading to pulmonary fibrosis. Histol Histopathol 1996;11:463–483.[Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200510-1648OCv1
174/5/557    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 Google Scholar
Google Scholar
Right arrow Articles by Kelly, M. M.
Right arrow Articles by Gauldie, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kelly, M. M.
Right arrow Articles by Gauldie, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Cell Mol. Biol.
Copyright © 2006 American Thoracic Society