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WNT7B in fibroblastic foci of idiopathic pulmonary fibrosis.
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MedLine Citation:
PMID:  22838404     Owner:  NLM     Status:  MEDLINE    
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial pneumonia causing a loss of respiratory surface area due to a proliferative fibrotic response involving hyperplastic, hypertrophic, and metaplastic epithelium, cystic honeycomb change, septal expansion, and variable inflammation. Wnt (wingless) signaling glycoproteins are known to be involved in lung development and tissue repair, and are up-regulated in patients with IPF. Based on previous qRT-PCR data showing increased Wnt7B in lungs of IPF patients, a systematic, quantitative examination of its tissue site distribution was undertaken.
METHODS: Tissue samples from the Lung Tissue Research Consortium (LTRC) of 39 patients diagnosed with mild to severe IPF/usual interstitial pneumonia (UIP) and 19 normal patients were examined for the immunolocalization of Wnt7B.
RESULTS: In normal lung, moderate Wnt7B reactivity was confined to airway epithelium, smooth muscle of airways and vasculature, and macrophages. IPF lung showed strong Wnt7B reactivity in fibroblastic foci, dysplastic airway and alveolar epithelium, and in highly discrete subepithelial, basement membrane-associated regions. All reactive sites were sized and counted relative to specific microscopic regions. Those in the subepithelial sites were found in significantly greater numbers and larger relative area compared with the others. No reactive sites were present in normal patient controls.
CONCLUSIONS: The results demonstrate Wnt7B to be expressed at high concentrations in regions of active hyperplasia, metaplasia, and fibrotic change in IPF patients. In this context and its previously established biologic activities, Wnt7B would be expected to be of potential importance in the pathogenesis of IPF.
Travis Meuten; Ariel Hickey; Katherine Franklin; Brian Grossi; Jeremy Tobias; Donna R Newman; Samuel H Jennings; Maria Correa; Philip L Sannes
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2012-07-28
Journal Detail:
Title:  Respiratory research     Volume:  13     ISSN:  1465-993X     ISO Abbreviation:  Respir. Res.     Publication Date:  2012  
Date Detail:
Created Date:  2012-10-24     Completed Date:  2013-04-08     Revised Date:  2013-07-12    
Medline Journal Info:
Nlm Unique ID:  101090633     Medline TA:  Respir Res     Country:  England    
Other Details:
Languages:  eng     Pagination:  62     Citation Subset:  IM    
Departments of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607, USA.
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MeSH Terms
Aged, 80 and over
Basement Membrane / pathology
Fibroblasts / pathology*
Idiopathic Pulmonary Fibrosis / metabolism*,  pathology*
Macrophages / pathology
Middle Aged
Muscle, Smooth / pathology
Respiratory Mucosa / pathology
Respiratory System / blood supply,  pathology
Severity of Illness Index
Wnt Proteins / analysis,  metabolism*
Grant Support
Reg. No./Substance:
0/WNT7B protein, human; 0/Wnt Proteins

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Journal Information
Journal ID (nlm-ta): Respir Res
Journal ID (iso-abbrev): Respir. Res
ISSN: 1465-9921
ISSN: 1465-993X
Publisher: BioMed Central
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Copyright ©2012 Meuten et al.; licensee BioMed Central Ltd.
Received Day: 25 Month: 5 Year: 2012
Accepted Day: 17 Month: 7 Year: 2012
Print publication date: Year: 2012
Electronic publication date: Day: 28 Month: 7 Year: 2012
Volume: 13 Issue: 1
First Page: 62 Last Page: 62
PubMed Id: 22838404
ID: 3479038
Publisher Id: 1465-9921-13-62
DOI: 10.1186/1465-9921-13-62

WNT7B in fibroblastic foci of idiopathic pulmonary fibrosis
Travis Meuten1 Email:
Ariel Hickey1 Email:
Katherine Franklin1 Email:
Brian Grossi1 Email:
Jeremy Tobias2 Email:
Donna R Newman1 Email:
Samuel H Jennings1 Email:
Maria Correa2 Email:
Philip L Sannes1 Email:
1Departments of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, 1060 William Moore Dr, Raleigh, NC 27607, USA
2Population Health and Pathology, enter for Comparative Medicine and Translational Research, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA


Idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP) is a debilitating disease characterized by a loss of normal respiratory architecture and replacement with a heterogeneous population of myofibroblast-like cells and excess of fibrous connective tissue restricted to the lung [1,2]. IPF arises from inflammation in the alveolar-capillary wall resulting in alveolar type I cell (AT1) loss and AT2 cell hyperplasia and subepithelial/interstitial fibrogenesis [3,4]. It has been suggested this represents an attempt to repair the pulmonary barrier following an injury to the respiratory surface [5]. The hallmark lesions are fibroblastic foci, signifying active disease, with a patchy mix of older fibrosis starting from the subpleural surface and along interlobular septa. Early lesions frequently appear highly cellular, with subepithelial fibroblastic foci adjacent to normal pulmonary architecture [6,7]. In IPF the lesions lead to end-stage fibrosis with minimal remaining pulmonary structure [6-8]. The proposed pathogenesis centers on dysregulation of epithelial repair in the form of hyperplastic and metaplastic AT2 cells interrelated with fibroproliferative lesions and aberrant epithelial differentiation, including epithelial to mesenchymal transition (EMT) [8,9].

The signaling pathways involved are only partially understood. It is known that there is increased expression of TGF-β and α-smooth muscle actin (α-SMA) in progressive lesions of IPF reflecting the transition of fibroblasts to myofibroblasts with proliferation and collagen maintenance modulated by wingless (Wnt) glycoproteins [10-12]. Mouse models indicate cooperation of TGF-β and Wnt signaling pathways in development, differentiation, and EMT [13]. Altered expression of Wnt ligands and one of their downstream targets, β-catenin, is evident in early IPF and bleomycin models of pulmonary fibrosis [14-16]. This can be explained in light of up-regulated TGF-β by its induction of LEF-1, which is a component of the canonical Wnt signaling pathway [13]. Chilosi, et al., found highly concentrated sites of β-catenin in myofibroblast-populated regions adjacent to airways [14]. This correlated with elevated mRNA expression and immunohistochemical reactivity of Wnts 1 & 3a in adjacent pulmonary epithelium in IPF patients [17]. Recent evidence shows that Wnt3a can activate β-catenin-mediated signaling and induce EMT in lung epithelial cells [18]. The collective views suggest that canonical Wnt signaling (β-catenin mediated) is up-regulated in fibrogenic conditions and may be causally involved in IPF [16-19].

Wnt glycoproteins have been found to be expressed at low levels in the normal lung and may be associated with epithelial turnover [17,20]. For example, Wnt7B mRNA expression in isolated normal AT2 cells was evident in low levels [17,21]. Wnt7b over-expression is thought to be a contributing factor to fibrogenesis in the murine kidney [22] and to be involved with procollagen production by lung fibroblasts [12]. Wnt7b was of special interest because of its role in mesenchymal proliferation and vascular development in the lung [23]. Based on supporting qRT-PCR data showing increased Wnt7B in lungs of IPF patients [17], a systematic, examination of its localization in a cohort of 39 IPF lungs was undertaken. Wnt7B expression was found in spindle cells and extracellular matrix of virtually all fibroblastic foci and in widely distributed, large numbers of discretely defined regions of the subepithelial basement membrane zone. These findings may provide useful clues relating to the pathogenesis of IPF and present novel, potential targets for detection and treatment.

Materials & methods

Tissue blocks of formalin-fixed lung tissue samples were obtained from the Lung Tissue Research Consortium (LTRC). The samples had previously been placed into three groups (see Table1): group 1, with forced vital capacities (FVCs) >80% (normal, or no specified major or minor diagnosis, n = 19); group 2, with FVCs between 50-80% [major final clinical diagnosis as interstitial lung disease (ILD, n = 20) and minor final clinical diagnosis as usual interstitial pneumonia (UIP)/idiopathic pulmonary fibrosis (IPF)]; and group 3, with FVCs <50% (major final clinical diagnosis of ILD and minor final clinical diagnosis as UIP/IPF, n = 19). No other patient identifiers were provided, and their anonymity and confidentiality were preserved. The study was approved by the North Carolina State University Institutional Review Board. Blocks were further randomized and sections stained with hematoxylin and eosin (H&E) and picrosirius red (PSR) or Movat’s pentachrome stain for collagen. H&E sections were examined by a Board Certified Pathologist to independently confirm/reclassify initial clinical diagnoses.

Sections were treated with citrate buffer for antigen retrieval and treated with a polyclonal goat anti-Wnt7B antibody [Santa Cruz Biotechnology, Inc. (sc26363, lot# I0205, Santa Cruz, CA)] at a 1:100 dilution overnight at 4°C, followed by peroxidase-labeled secondary antibodies (Dako LSAB+, Dako Laboratories, Carpinteria,CA) and Nova Red (Vector Laboratories, Burlingame, CA). The Wnt7B antibody recognizes both precursor and mature forms of human origin. Control samples substituted normal goat serum for the primary antibody, or were pre-treated with 5% testicular hyaluronidase (Sigma, Type 1-S, St. Louis, MO) for 30 minutes at 37°C to release Wnt7B bound to extracellular matrices [24], or treated with a competitive antibody-binding Wnt7B peptide to the primary antibody incubation. Sections were counterstained with methylene blue. Selected sequential and non-sequential serial sections were immunostained with anti-human smooth muscle actin.

Analysis of Reactive Sites

The entirety of each section was systematically evaluated at 200X magnification through a calibrated ocular grid, and the size of each Wnt7B reactive site was measured. They were divided into three major size categories: <50 μm2, 50–100 μm2, and >100 μm2; while their histologic sites were separated into one of three regional designations: 1) epithelium and underlying fibroblasts with or without extracellular matrix (EF), 2) subepithelial fibroblasts and ECM (SE) without epithelial staining (included most fibroblastic foci), and 3) interstitial fibroblasts and ECM (I) that were not directly subjacent to an epithelial surface. The number of sites corresponding to the respective categories was recorded. The total surface area, as defined by the external border of each tissue section, was determined using ImageJ (National Institutes of Health, Bethesda, MD), and the percent of the total area of each reactive site category relative to the total section surface area was calculated (see Table1).

Statistical analysis

Comparisons of the descriptive statistics of reactive sites were made using Analysis of Variance or Kruskal-Wallis non-parametric test for histologic locations in the same reactive site groups and within lung capacity category using SAS software (Cary, NC) or Minitab (Six Sigma, State College, PA). For example, for patients in FVC 50-80%, comparisons were made for reactive sites >100 (μm2) for EF, SE, and I locations. Statistical significance was set at an alpha value of ≤ 0.05.


Lungs with normal pulmonary architecture (group 1) had weak to moderate Wnt7B immunoreactivity in the cytoplasm of airway epithelium (ciliated cells) and smooth muscle of airways and arteries (Figure1a and inset). Much weaker Wnt7B reactivity was evident in the cytoplasm of AT2 cells (Figure1a inset), alveolar macrophages, and endothelial cells. Lungs originally categorized with ILD and UIP/IPF (by the LTRC) had intense and discrete immunoreactive sites for Wnt7B that varied in size and location. The largest sites correlated directly with easily identifiable fibroblastic foci characteristic of IPF, where staining was particularly intense in the extracellular matrix (Figures 1b1e2b2c2e). All sites, regardless of size and location, were unreactive with normal goat serum controls (Figure1c). Pretreatment of sections with 5% hyaluronidase prior to immunostaining for Wnt7B completely attenuated the intense extracellular matrix reactive sites seen in parallel sections, while most cellular reactivity was retained and somewhat intensified (Figure1d). This was particularly true of smooth muscle cells (data not shown). This increased cellular staining is likely due to the antibody detection of intracellular and cell surface-associated forms of the antigen, which is glycosylated [25], and made more available by the digestion procedure.

Simultaneous treatment of sections with Wnt7B antibody and its competitive binding Wnt7B peptide during the staining procedure resulted in significant reduction in all reactive sites in parallel sections, particularly in sites with the most intense immunoreactivity (Figure1e vs. 1f), confirming the specificity of the staining.

Even at lower magnifications, the Wnt7B reactive sites were readily detectable, especially the fibroblastic foci (Figure2a). Reactive sites were consistently found in one of three regional dispositions according to the criteria described above: EF, SE (which included most fibroblastic foci), and I (Figure2b). Sequential and non-sequential serial sections were used to compare Wnt7B stained slides with those stained with H&E, picrosirius red (PSR), Movat’s pentachrome, or immunohistochemistry for smooth muscle actin. Fibroblastic foci were easily detected by their large size (typically >100 μm2) and strong extracellular reactivity for Wnt7B, with most found adjacent to thickened airways (Figure2b-c) and thickened interstitium in alveolar regions (1e, 2e). They correlated well with sequential/non-sequential serial sections stained with H&E (data not shown) and pentachrome (2d, 2f). The red reactivity of collagen with PSR was very strong throughout IPF lungs, especially in highly thickened areas of mature collagen and thickened basal laminae (data not shown). Fibroblastic foci were easily identifiable, mainly by virtue of their lighter staining, immature collagen content (as shown with the Movat’s pentachrome), which gave it a finely layered appearance (data not shown).

Wnt7B immunoreactivity was found both intra- and extracellularly. Without hyaluronidase digestion, the extracellular reactivity was discrete and intense and found in the ECM regions of fibroblastic foci (Figures 1b, 1e, 2b-c, 2f), subepithelial ECM (Figure2a-b, 3b), and interstitium (Figure2b) not adjacent to epithelium. The sizes of the reactive sites varied widely, with fibroblastic foci having the greatest individual reactive areas (often >1600 μm2; Figures 1b, 2c, 2b-c, 2e) and the SE regions being the most numerous and smallest in individual reactive area (Figure2b, 3a-d). These smaller sites were not distinguishable by any morphologic features or special staining characteristics (data not shown). Intracellular reactivity was very intense in some epithelia, especially that considered hyper- or metaplastic (Figure3a), and in fibroblasts of fibroblastic foci, especially the larger SE sites (Figure3c) and smaller SE sites (Figure3d). In airways, basal cells were often reactive (Figure3d), as were some ciliated cells (data not shown). Mesothelium was generally positive, but mesothelial immunoreactivity was most intense when overlying areas of interstitial fibrosis (Figure2g). Macrophages were variably reactive for Wnt7B, while other inflammatory cells present in IPF/UIP lungs were uniformly negative (data not shown). Many SE sites reflected staining of portions of basal lamina (Figures 3b-d) and/or strong staining of fibroblasts within the subepithelial fibroblast layer (Figure3d). The fibroblastic component of Wnt7B-positive fibroblastic foci (Figure3d-e) was uniformly positive for smooth muscle actin (Figure3f), reflective of the myofibroblast phenotype.

Patients with FVC > 80% had few quantifiable Wnt7B reactive sites (Figure4a) compared to patients with functional diagnoses of IPF/UIP [<80% (Figures 4b-c)]. There were insufficient reactive sites for the EF category to compare to corresponding reactive SE and I regions. Despite an outlier (defined as greater than 2 standard deviations from the mean) for the SE region <50 μm2 category, the reactive sites for locations SE and I were not statistically different, given that the median value was 0 for all reactive sites by location. For patients with FVC 50-80% and FVC <50%, reactive site size means (in μm2), standard deviations, and minimum, median, and maximum sizes of reactive sites (in μm2) are presented in Tables 2 and 3. For patients with FVC 50-80% and FVC <50%, the means of the number of each of the reactive site sizes for the SE location were statistically greater than that of those for EF and I locations (p < 0.05).


The initiating cause(s) underlying the pathogenesis of idiopathic pulmonary fibrosis (IPF) is unknown. Much attention has focused on a failed repair of alveolar epithelium, and its impact on subsequent loss of pulmonary architecture. This failure manifests as the inability of epithelial surfaces to proliferate and differentiate effectively resulting in hyperplasia, metaplasia, and/or transdifferentiation into myofibroblast-like cells [26]. Exactly how these dysplastic events culminate, independently or collectively, in the formation of one of the hallmarks of IPF, the fibroblastic focus, is not clear. Studying the roles that specific signaling pathways play in the complex processes involved in the formation of fibroblastic foci has led to a greater understanding of important growth factors, such as TGF-β, that not only promote the deposition of excessive collagen but mediate the process of EMT as well [10]. TGF-β has been immunohistochemically localized to alveolar epithelium and extracellular matrix of IPF lungs [27] and is within fibroblastic foci [28,29]. Fibroblastic foci are also rich in its downstream nuclear targets like phospho-Smad 2/3 [28] and relevant proteins, such as cysteine-rich protein 1 (CRP-1), which is important in smooth muscle cell differentiation [30]. Relatedly, activated β-catenin has been shown to be up-regulated in the nuclei of myofibroblasts of fibroblastic foci of IPF patients [14], and alveolar epithelial responses to TGF-β involve alpha3 integrin for β-catenin phosphorylation and formation of a β-catenin/p-Smad2 complex resulting in initiation of EMT [31]. More specifically, TGF-β-stimulated Smad3 has recently been shown to form a complex with β-catenin and CREB-binding protein in regulation of α-smooth muscle actin, a cytologic signature of EMT [32]. SPARC (secreted protein acidic rich in cysteine), an extracellular matrix component abundant in fibroblastic foci [33], should also be noted, as it has been shown to activate AKT, inhibit GSK-3β, and activate β-catenin, resulting in an anti-apoptotic phenotype [34], another characteristic of the IPF myofibroblast. [10-12].

The finding of strong Wnt7B immunoreactivity in the fibroblastic focus of UIP/IPF lungs would seem to fit readily with the above discussion on canonical Wnt signaling involving β-catenin. The demonstration of Wnt7B in dysplastic airway and alveolar epithelium and in myofibroblasts of the fibroblastic foci closely correlates with the site localization of active β-catenin in IPF lungs [14]. However, that study did not report active β-catenin in fibroblasts/myofibroblasts in regions other than fibroblastic foci. Wnt7B localization, demonstrated here, was also clearly defined in a large number of smaller sites (<50 μm2) that, like fibroblastic foci, had both cellular and extracellular matrix components. The loss of extracellular Wnt7B immunoreactivity after hyaluronidase digestion supports the matrix-associated localization, which is not surprising, as Reichsman, et al., [24] have demonstrated that most secreted Wnts (approximately 83%) are bound to the cell surface and surrounding extracellular matrix through specific, non-covalent interactions. The localization of Wnt7B in fibroblastic foci and a large number of smaller subepithelial sites position it well for influencing the activity of the underlying interstitium. In normal lung development, murine Wnt7b has been shown to be exclusively expressed in epithelial cells and regulated by TTF-1, GATA-6, and FoxA2 [35]. In the embryo, it has been demonstrated to stimulate epithelial and mesenchymal proliferation, likely through its activation of BMP-4 and Id2[36]. Further, Wnt7b/β-catenin signaling regulates a program of mesenchymal cell differentiation and proliferation that is necessary for smooth muscle cell development in cooperation with tenascin-C and involving PDGFR-alpha and PDGFR-beta [37]. It may not be surprising, then, that similar genetic programs are reactivated in some form in an adult disease such as IPF, in which tenascin-C is heavily expressed in the matrix of fibroblastic foci [33] where myofibroblasts expressing PDGFRs are located [38]. Coupled with the strong expression of Wnt7B in hyperplastic/metaplastic epithelium and fibroblastic foci reported here, these observations support previous conclusions that epithelium in IPF may be responsible for aberrant activation of Wnt signaling, such as that of Wnt7B, in adjacent mesenchyme, leading to damage to the lung and fibrosis [19].

The notion that Wnt7B may have a potentially contributive if not significant role in the development and/or progression of IPF draws on previous assumptions that the fibroblastic foci are central to the disease process and its prognosis [39,40]. The fibroblastic foci were the most dramatic sites of Wnt7B localization observed in the IPF lung specimens. However, when systematically categorized and analyzed relative to location and size, the Wnt7B reactive sites of large fibroblastic foci were not the most numerous nor did they consume the greatest percentage of total tissue section area, regardless of disease severity according to % FVC, assuming the section samples correctly reflected the overall extent of fibrotic change of the whole lung (see Figure4). This distinction belonged to the small subepithelial (SE) sites <50 μm2 (Figures 1d2a3b-d). These unique reactive sites had no other morphological features that were specifically distinguishing other than their discrete Wnt7B immunoreactivity. In parallel sections, the small SE sites, like the larger fibroblastic foci, consistently contained abundant cells expressing the myofibroblast phenotype, as indicated by α-smooth muscle actin immunoreactivity (Figure3f), but by virtue of their Wnt7B reactivity, clearly represented a subpopulation of these cells compared with the overall total myofibroblast population in IPF lungs. While it is not clear from the current data that there are any connections between the Wnt7B positive reactivity of the fibroblastic foci and the more numerous, smaller SE sites, Cool, et al., [41] demonstrated with three-dimensional reconstruction of pentachrome-stained sections that fibroblastic foci of UIP form a complex, interconnected network that extends from the pleura into the parenchyma. Perhaps the strong reactivity for Wnt7B within fibroblastic foci, shown here to correlate precisely with pentachrome reactivity, along with the foci’s complement of established pro-fibrogenic components constitute the expansion unit of IPF, and the small Wnt7B-positive, subepithelial (SE) sites are the leading edge of this process. The location of the small SE sites could portend the epithelial-fibroblast cross-talk often involving Wnt signaling and known to be important determinants of the fibrogenic events characteristic of IPF [26]. The regionally confined localization of Wnt7B differs from that of Wnt5A, which is strongly expressed in the majority of fibroblasts derived from UIP patients (i.e., almost all of the remaining fibroblasts), and shown to signal through non-canonical pathways, promote proliferation, and prevent apoptosis [42]. It raises the possibility that Wnt7B and Wnt5A, along with TGF-β, SPARC, and tenascin-C, work in some coordinated or concerted fashion to modulate fibroblast/myofibroblast activities in adjacent and/or different anatomic regions of IPF lungs. Extensive and more detailed studies are currently underway to help develop a better understanding of this complex process.


These observations draw attention to a specific Wnt signaling ligand, Wnt7B, which by virtue of its established roles in epithelial and mesenchymal proliferation and differentiation, procollagen production, and enhanced gene expression in IPF, would be expected to act as a significant contributor to the pathogenesis of IPF. This is supported not only by the strong expression of Wnt7B in fibroblastic foci but also by the numerous, small subepithelial sites that may represent early stages of developing fibroblastic foci as part of a larger, expanding network of fibrogenic tissue.

Competing interests

There are no competing interests to report by any of the authors.

Authors’ contributions

TM – performed immunostaining, prepared photographic images, drafting and revision of manuscript; AH - performed immunostaining, KF - developed protocol staining and performed immunostaining; BG - performed immunostaining; JT – developed immunostainig protocol; DN - interpretation of data, drafting and revision of manuscript; SJ – analysis and interpretation of data, manuscript revision; MC - analysis and interpretation of data, statistical analysis; PS – conception and design of study, analysis and interpretation of data, preparation of photographic images, drafting of manuscript and revision; all authors read and approved of the final manuscript.


This investigation was supported by PHS grants HL44497, HL95411, North Carolina State University College of Veterinary Medicine, and the State of North Carolina. The authors wish to thank Thomas Sporn, M.D., Department of Pathology, Duke University Medical Center, Durham, NC, for his guidance and diagnostic support, and Laura Shewmon, Department of Population Health and Pathology, College of Veterinary Medicine, North Carolina State University for her technical contribution to this study.

Green FH,Overview of pulmonary fibrosisChestYear: 2002122334S339S10.1378/chest.122.6_suppl.334S12475811
Schwartz DA,Helmers RA,Galvin JR,Van Fossen DS,Frees KL,Dayton CS,Burmeister LF,Hunninghake GW,Determinants of survival in idiopathic pulmonary fibrosisAm J Respir Crit Care MedYear: 19941494504548306044
Strieter RM,Con: Inflammatory mechanisms are not a minor component of the pathogenesis of idiopathic pulmonary fibrosisAm J Respir Crit Care MedYear: 200216512061207 discussion 1207–8. 10.1164/rccm.220205511991867
Strieter RM,Pathogenesis and natural history of usual interstitial pneumonia: the whole story or the last chapter of a long novelChestYear: 2005128526S532S10.1378/chest.128.5_suppl_1.526S16304243
Konigshoff M,Eickelberg O,WNT signaling in lung disease: a failure or a regeneration signal?Am J Respir Cell Mol BiolYear: 201042213110.1165/rcmb.2008-0485TR19329555
Katzenstein AL,Myers JL,Idiopathic pulmonary fibrosis: clinical relevance of pathologic classificationAm J Respir Crit Care MedYear: 1998157130113159563754
Katzenstein AL,Zisman DA,Litzky LA,Nguyen BT,Kotloff RM,Usual interstitial pneumonia: histologic study of biopsy and explant specimensAm J Surg PatholYear: 2002261567157710.1097/00000478-200212000-0000412459623
Kapanci Y,Desmouliere A,Pache JC, Redard MGabbiani G: Cytoskeletal protein modulation in pulmonary alveolar myofibroblasts during idiopathic pulmonary fibrosis. Possible role of transforming growth factor beta and tumor necrosis factor alpha. Am J Respir Crit Care MedYear: 199515221632169
Adamson IY,Hedgecock C,Bowden DH,Epithelial cell-fibroblast interactions in lung injury and repairAm J PatholYear: 19901373853921696785
Broekelmann TJ,Limper AH,Colby TV,McDonald JA,Transforming growth factor beta 1 is present at sites of extracellular matrix gene expression in human pulmonary fibrosisProc Natl Acad Sci USAYear: 1991886642664610.1073/pnas.88.15.66421862087
Scotton CJ,Chambers RC,Molecular targets in pulmonary fibrosis: the myofibroblast in focusChestYear: 20071321311132110.1378/chest.06-256817934117
Salazar KD,Lankford SM,Brody AR,Mesenchymal stem cells produce Wnt isoforms and TGF-beta1 that mediate proliferation and procollagen expression by lung fibroblastsAm J Physiol Lung Cell Mol PhysiolYear: 2009297L1002L101110.1152/ajplung.90347.200819734317
Attisano L,Labbe E,TGFbeta and Wnt pathway cross-talkCancer Metastasis RevYear: 200423536115000149
Chilosi M,Poletti V,Zamo A,Lestani M,Montagna L,Piccoli P,Pedron S,Bertaso M,Scarpa A,Murer B,Cancellieri A,Maestro R,Semenzato G,Doglioni C,Aberrant Wnt/beta-catenin pathway activation in idiopathic pulmonary fibrosisAm J PatholYear: 20031621495150210.1016/S0002-9440(10)64282-412707032
Douglas IS,Diaz del Valle F, Winn RA, Voelkel NF: Beta-catenin in the fibroproliferative response to acute lung injuryAm J Respir Cell Mol BiolYear: 20063427428516272459
Willert K,Nusse R,Beta-catenin: a key mediator of Wnt signalingCurr Opin Genet DevYear: 199889510210.1016/S0959-437X(98)80068-39529612
Konigshoff M,Balsara N,Pfaff EM,Kramer M,Chrobak I,Seeger W,Eickelberg O,Functional Wnt signaling is increased in idiopathic pulmonary fibrosisPLoS OneYear: 20083e214210.1371/journal.pone.000214218478089
van der Velden JL,Guala AS,Leggett SE,Sluimer J,Badura EC,Janssen-Heininger YM,Induction of a Mesenchymal Expression Program in Lung Epithelial Cells by Wnt/beta-catenin Requires the Presence of c-Jun N-Terminal Kinase 1Am J Respir Cell Mol BiolYear: 2012 Published ahead of print on March 2012. 10.1165/rcmb.2011-029
Morrisey EE,Wnt signaling and pulmonary fibrosisAm J PatholYear: 20031621393139710.1016/S0002-9440(10)64271-X12707021
Winn RA,Marek L,Han SY,Rodriguez K,Rodriguez N,Hammond M,Van Scoyk M,Acosta H,Mirus J,Barry N,Bren-Mattison Y,Van Raay TJ,Nemenoff RA,Heasley LE,Restoration of Wnt-7a expression reverses non-small cell lung cancer cellular transformation through frizzled-9-mediated growth inhibition and promotion of cell differentiationJ Biol ChemYear: 2005280196251963410.1074/jbc.M40939220015705594
Apparao KB,Newman DR,Zhang H,Khosla J,Randell SH,Sannes PL,Temporal changes in expression of FoxA1 and Wnt7A in isolated adult human alveolar epithelial cells enhanced by heparinAnat Rec (Hoboken)Year: 201029393894610.1002/ar.2080520503388
He W,Dai C,Li Y,Zeng G,Monga SP,Liu Y,Wnt/beta-catenin signaling promotes renal interstitial fibrosisJ Am Soc NephrolYear: 20092076577610.1681/ASN.200806056619297557
Shu W,Jiang YQ,Lu MM,Morrisey EE,Wnt7b regulates mesenchymal proliferation and vascular development in the lungDevelopmentYear: 20021294831484212361974
Reichsman F,Smith L,Cumberledge S,Glycosaminoglycans can modulate extracellular localization of the wingless protein and promote signal transductionJ Cell BiolYear: 199613581982710.1083/jcb.135.3.8198909553
Smolich BD,McMahon JA,McMahon AP,Papkoff J,Wnt family proteins are secreted and associated with the cell surfaceMol Biol CellYear: 19934126712758167409
Coward WR,Saini G,Jenkins G,The pathogenesis of idiopathic pulmonary fibrosisTher Adv Respir DisYear: 2010436738810.1177/175346581037980120952439
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 studyAm J Respir Cell Mol BiolYear: 1996141311388630262
Jonigk D,Theophile K,Hussein K,Bock O,Lehmann U,Bockmeyer CL,Gottlieb J,Fischer S,Simon A,Welte T,Maegel L,Kreipe H,Laenger F,Obliterative airway remodelling in transplanted and non-transplanted lungsVirchows ArchYear: 201045736938010.1007/s00428-010-0949-x20632031
Lomas NJ,Watts KL,Akram KM,Forsyth NR,Spiteri MA,Idiopathic pulmonary fibrosis: immunohistochemical analysis provides fresh insights into lung tissue remodelling with implications for novel prognostic markersInt J Clin Exp PatholYear: 20125587122295148
Jarvinen PM,Myllarniemi M,Liu H,Moore HM,Lepparanta O,Salmenkivi K,Koli K,Latonen L,Band A,Laiho M,Cysteine-rich protein 1 is regulated by transforming growth factor-beta1 and expressed in lung fibrosisJ Cell PhysiolYear: 201122762605261221882188
Kim KK,Wei Y,Szekeres C,Kugler MC,Wolters PJ,Hill ML,Frank JA,Brumwell AN,Wheeler SE,Kreidberg JA,Chapman HA,Epithelial cell alpha3beta1 integrin links beta-catenin and Smad signaling to promote myofibroblast formation and pulmonary fibrosisJ Clin InvestYear: 200911921322419104148
Zhou B,Liu Y,Kahn M,Ann DK,Han A,Wang H,Nguyen C,Flodby P,Zhong Q,Krishnaveni MS,Liebler JM,Minoo P,Crandall ED,Borok Z,beta-gJ Biol ChemYear: 2012201220122012
Kuhn C,Mason RJ,Immunolocalization of SPARC, tenascin, and thrombospondin in pulmonary fibrosisAm J PatholYear: 1995147175917697495300
Chang W,Wei K,Jacobs SS,Upadhyay D,Weill D,Rosen GD,SPARC suppresses apoptosis of idiopathic pulmonary fibrosis fibroblasts through constitutive activation of beta-cateninJ Biol ChemYear: 20102858196820610.1074/jbc.M109.02568420061390
Weidenfeld J,Shu W,Zhang L,Millar SE,Morrisey EE,The WNT7b promoter is regulated by TTF-1, GATA6, and Foxa2 in lung epitheliumJ Biol ChemYear: 2002277210612107010.1074/jbc.M11170220011914369
Rajagopal J,Carroll TJ,Guseh JS,Bores SA,Blank LJ,Anderson WJ,Yu J,Zhou Q,McMahon AP,Melton DA,Wnt7b stimulates embryonic lung growth by coordinately increasing the replication of epithelium and mesenchymeDevelopmentYear: 20081351625163410.1242/dev.01549518367557
Cohen ED,Ihida-Stansbury K,Lu MM,Panettieri RA,Jones PL,Morrisey EE,Wnt signaling regulates smooth muscle precursor development in the mouse lung via a tenascin C/PDGFR pathwayJ Clin InvestYear: 20091192538254910.1172/JCI3807919690384
Vuorinen K,Ohlmeier S,Lepparanta O,Salmenkivi K,Myllarniemi M,Kinnula VL,Peroxiredoxin II expression and its association with oxidative stress and cell proliferation in human idiopathic pulmonary fibrosisJ Histochem CytochemYear: 20085695195910.1369/jhc.2008.95180618606608
Enomoto N,Suda T,Kato M,Kaida Y,Nakamura Y,Imokawa S,Ida M,Chida K,Quantitative analysis of fibroblastic foci in usual interstitial pneumoniaChestYear: 2006130222910.1378/chest.130.1.2216840378
Flaherty KR,Colby TV,Travis WD,Toews GB,Mumford J,Murray S,Thannickal VJ,Kazerooni EA,Gross BH,Lynch JP,Martinez FJ,Fibroblastic foci in usual interstitial pneumonia: idiopathic versus collagen vascular diseaseAm J Respir Crit Care MedYear: 20031671410141510.1164/rccm.200204-373OC12615630
Cool CD,Groshong SD,Rai PR,Henson PM,Stewart JS,Brown KK,Fibroblast foci are not discrete sites of lung injury or repair: the fibroblast reticulumAm J Respir Crit Care MedYear: 200617465465810.1164/rccm.200602-205OC16799077
Vuga LJ,Ben-Yehudah A,Kovkarova-Naumovski E,Oriss T,Gibson KF,Feghali-Bostwick C,Kaminski N,WNT5A is a regulator of fibroblast proliferation and resistance to apoptosisAm J Respir Cell Mol BiolYear: 20094158358910.1165/rcmb.2008-0201OC19251946


[Figure ID: F1]
Figure 1  

Section of normal human lung treated for the immunohistochemical localization of Wnt7B. Figure1a at low power, airway (A) epithelial cells and smooth muscle (double arrows) surrounding a nearby vessel (V) exhibit reactivity for Wnt7B. Inset of the airway shows Wnt7B reactivity in ciliated cells (solid arrowtip), AT2 cell (single arrow), and smooth muscle (double arrows). The airway insert also shows and non-reactive non-ciliated bronchiolar cell (solid arrowhead). Methylene blue counterstain. Bar = 100 μm; inset Bar = 30 μm. Figure1b, Section of UIP/IPF lung treated for the immunohistochemical localization of Wnt7B. Two large fibroblastic foci (FF) demonstrate strong extracellular reactivity. Bar = 100 μm. Figure1c, Serial section of UIP/IPF lung in Figure1b treated with non-immune serum instead of the Wnt7B-specific antibody. Fibroblastic foci (FF) lack selective staining for the antigen, but show light metachromasia with the methylene blue counterstain. Bar = 100 μm. Figure1d, Serial section of UIP/IPF lung in Figure1b treated for the immunohistochemical localization of Wnt7B after being exposed for 30 minutes to hyaluronidase digestion. The same fibroblastic foci (FF) have lost the strong, extracellular reactivity seen in Figure1b, while there is enhanced cellular reactivity throughout the tissue, presumptively due to unmasking of intracellular, glycosylated Wnt7B. Methylene blue counterstain. Bar = 100 μm. Figure1e-f, Serial sections of UIP/IPF lung treated for the immunohistochemical localization of Wnt7B; the first (e) without and second (f) with the antigenic blocking peptide simultaneously with the Wnt7B-specific antibody. The two positive fibroblastic foci (FF) in (e) show diminished selective staining for Wnt7B in (f). Methylene blue counterstain. Bar = 100 μm.

[Figure ID: F2]
Figure 2  

Low power magnification of a section of UIP/IPF lung treated for the immunohistochemical localization of Wnt7B. Fibroblastic foci are easy to identify (large arrows), as are even small reactive sites (small arrows). Methylene blue counterstain. Bar = 600 μm. Figure2b, Section of UIP/IPF lung treated for the immunohistochemical localization of Wnt7B demonstrating the three categories designated for sizing and quantitation. Preliminary histopathologic evaluation of the slides determined that all reactive sites were confined to one of three characteristic types: epithelial + fibroblast + extracellular matrix (EF), subepithelial (SE, fibroblast + extracellular matrix), or interstitial (I, not adjacent to epithelial surfaces). Methylene blue counterstain. Bar = 100 μm. Figure2c-f, Serial sections of UIP/IPF lung treated for the immunohistochemical localization of Wnt7B (c, e) or Movat’s pentachrome (d, f) in airway (c, d) and alveolar (e. f) regions. The same fibroblastic foci (FF) stain clearly with both treatments. Methylene blue counterstain. Bar = 100 μm. Figure2g, Section of UIP/IPF lung treated for the immunohistochemical localization of Wnt7B demonstrating reactivity of the mesothelial surface (arrows) and the subjacent cells and thickened interstitium. Methylene blue counterstain. Bar = 100 μm.

[Figure ID: F3]
Figure 3  

Sections of UIP/IPF lung treated for the immunohistochemical localization of Wnt7B. Figure3a demonstrates staining of hypertrophic/metaplastic alveolar epithelial cells. Figure3b demonstrates extensive subepithelial (SE, not an FF) site reactivity (arrows) around one side of a small airway. Figure3c demonstrates reactivity within a fibroblastic focus (FF) and adjacent SE sites (arrows). The inset at higher magnification of the fibroblastic foci demonstrates the extracellular matrix and cellular (arrow) nature of the reactivity. Figure3d demonstrates extensive subepithelial (SE) site reactivity (arrows) around two small airways, the apparent reactivity of presumptive basal cells (arrows) within the respective airways. The inset at higher magnification shows presumptive, reactive fibroblasts embedded within reactive matrix (arrowheads). Methylene blue counterstain. Bars = 50 μm; inset bars = 30 μm.

[Figure ID: F4]
Figure 4  

Distribution of the total Wnt7B reactivity at the EF, SE, and I site categories for patients with FVCs >80% (normal), 50-80% (intermediate impairment), or <50% (severe impairment). Normal patients had minimal Wnt7B reactivity, most with a median total area reactivity of “0” (a). Patients with impaired FVCs had many more reactive sites than controls, although those with intermediate and severe impairment were not different (see Tables 2 and 3). Notably, the median of the mean total of reactivity was greatest in the SE sites that were <50 μm² (b-c).

[TableWrap ID: T1] Table 1 

Patient group data summary and total Wnt7B area reactivity

Patient ID Area Total μm2/mm2 Path Diagnosis FVC > 80% Patient ID Area Total μm2/mm2 Path Diagnosis FVC > 50% < 80% Patient ID Area Total μm2/mm2 Path Diagnosis FVC < 50%
Mild IF
Mild IF
Mild IF
Mild IF
Severe IF
Mild emphysema
Mild IF
Mild IF
Severe IF
Mild IF
Mild IF
Mild emphysema
Mild IF
Septal thickening
Mild IF
Mild IF
Mild emphysema
A telectasis/thickening
Mild IF
stdev 0.097448895   stdev 0.808903868   stdev 0.71592836  

Legend: IF – interstitial fibrosis; IPF – idiopathic pulmonary fibrosis; UIP – usual interstitial pneumonia.

[TableWrap ID: T2] Table 2 

Patients with FVC > 50% < 80%

Site/size Mean StDev Min Med Max
EF > 100
EF >50 < 100
EF <50
SE >100
SE >50 < 100
SE <50
I >100
I >50 < 100
I <50 0.1044 0.152 0 0.0458 0.6613

[TableWrap ID: T3] Table 3 

Patients with FVC < 50%

Site/size Mean StDev Min Med Max
EF >100
EF >50 < 100
EF <50
SE >100
SE >50 < 100
SE <50
I >100
I >50 < 100
I <50 0.093 0.1135 0 0.0671 0.4284

* the means of the number of each of the reactive site sizes for the SE location were statistically greater than that of those for EF and I locations (p < 0.05).

Article Categories:
  • Research

Keywords: Myofibroblasts, Alveolar epithelium, Interstitial lung disease.

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