Cigarette smoke–induced lung disease presents a morphologic contradiction in that the small airways become fibrotic but the parenchyma becomes emphysematous over time. To examine the mechanisms behind these phenomena, we exposed mice to cigarette smoke for up to 6 months and isolated small airways from histologic sections by laser capture microdissection. We then removed residual airway tissue and vessels, and collected the remaining parenchymal tissue. Gene expression of 13 fibrogenic growth/signaling factors (particularly TGF-β–related genes), matrix proteins, or enzymes involved in matrix production was examined by real-time RT-PCR. Combining present and previously published data from our laboratory, in the airways over the long term there was a sustained and marked increase in expression of almost all of these genes. By contrast, in the parenchyma, expression of most genes was elevated at 2 and 24 hours after initial exposure, and all were elevated at 1 month; but by 6 months, when emphysema was present, most genes (9/13) were either at control values or down-regulated below control. At 3 months, several genes that were considerably elevated at 1 month were back to control levels, suggesting that loss of the parenchymal response precedes the development of emphysema. We conclude that with smoke exposure the airways demonstrate an ongoing profibrotic/proelastogenic response and the parenchyma a generally anti-fibrotic/anti-elastogenic response, but one that develops only with long-term exposure to smoke. These observations support the idea that the parenchyma largely fails to repair smoke-induced matrix damage, but this phenomenon is a relatively late event.
There is no corresponding “classic” theory of small airway remodeling. Rather, most investigators have assumed that it also is driven by the smoke-induced inflammatory response (3). We have recently shown, however, using laser capture microdissection (LCM) and real-time RT-PCR of isolated small airways, that in fact small airway remodeling appears to be mediated through smoke-induced growth factor production in the airway wall, and that the role of inflammatory cells is unclear (4).
These observations raise the crucial question of why the parenchyma is destroyed in smokers rather than becoming fibrotic in the same fashion as the small airways. A variety of theories have been proposed to account for this phenomenon (5, and see Discussion). One hypothesis, taken in its broadest sense, is that, as opposed to the small airways, the lung parenchyma in smokers is unable to repair in the normal fashion, and in particular is unable to replace matrix damaged directly or indirectly by cigarette smoke (6). Another hypothesis has been termed “failure of lung maintenance,” and suggests that smoke induces apoptosis of lung structural cells and that this leads to emphysema (7). Both these hypotheses imply that in the parenchyma there is a failure to up-regulate expression of genes required for new matrix production after cigarette smoke–induced damage.
One way to examine the question of whether the parenchyma fails to repair smoke-induced damage is to look at production of matrix proteins and the co-factors required for their function, but there is little information about the changes in gene expression of matrix proteins or co-factors in the parenchyma after smoke exposure. Several authors have reported microarray analysis of emphysematous human lung parenchyma (8–11), an approach that could shed light on the problem, but these studies have produced remarkably inconsistent results with almost no overlap in genes reported to be up- or down-regulated (11). In part this problems stems from considerable differences in case selection such as use of lungs from patients at different GOLD stages in different reports. In addition, these studies have sampled parenchyma as a whole. A recent article analyzing whole lung microarray expression in smoke-exposed rats noted decreases in procollagen type 1 and tropoelastin gene expression by microarray analysis at 6 months (12; C. Stevenson, personal communication). However, understanding what is going on in the lung in chronic obstructive pulmonary disease (COPD) requires separately examining the airways and the parenchyma, since, as noted above, these two compartments react to smoke in quite different ways.
In this article we used microdissection techniques to compare the responses of the small airways and airway/vessel-free parenchyma over time in mice exposed to smoke for up to 6 months, and in particular to look at whether the parenchyma fails to repair smoke-induced damage. Combining our current results with previously published data from our laboratory, we show that, initially, both the airways and parenchyma up-regulate expression of genes that control matrix production and content, but that with continuing smoke exposure, the parenchyma no longer responds or even down-regulates most of these genes.
Animals used in this study were C57Bl/6 mice (Charles River, Montreal, PQ, Canada) that were exposed either once to the whole smoke of four 2R1 Kentucky Research Cigarettes, or on a daily basis to three 2R1 cigarettes 5 days per week for up to 6 months. The animals used for the 2-hour, 24-hour, 1-month, and 6-month time points were the same animals reported in Reference 4, in which we described the changes in gene expression of procollagen type 1, TGF-β1, connective tissue growth factor (CTGF), and platelet-derived growth factor (PDGF)-A and -B in isolated small airways (bronchioles). Additional genes and sites (airway versus parenchyma) were examined in this study. Additional animals were exposed to smoke for 3 months. All animal studies were approved by the University of British Columbia Animal Care Committee.
For the single smoke exposure groups, animals were killed at 2 and 24 hours after starting smoke exposure. For the chronic exposures, animals were exposed to smoke for 1 month, 3 months, or 6 months and killed 24 hours after the last smoke exposure.
After killing, the lungs were removed and inflated with either cold 100% ethanol for LCM, or (for morphometry and immunohistochemistry) with 1% low-melting-point agarose at 25 cm H2O pressure followed by fixation in formalin, then sectioned, dehydrated, and embedded in paraffin (13). Tissue blocks to be used for LCM were stored at 4°C. For calculation of airspace size (mean linear intercept), histologic sections were prepared from four control and four smoke-exposed animals; airspace size was measured using the Image Pro (Silver Springs, MD) system, with a 130-line grid with a total line length of 1.25 mm from which intercepts and points were derived. We analyzed 15 randomly acquired photographs from each animal. Mean linear intercept was calculated using standard morphometric equations (14).
LCM was performed on 5-μm-thick histologic sections using an Arcturus Pixcell II (Arcturus, Mountain View, CA) LCM apparatus. Sections were stained with the Arcturus stain. For the present study we cut new sections from the same blocks used in Reference 4, or cut sections from the blocks of the animals exposed to smoke for 3 months, and did new LCMs, collecting small airways (membranous bronchioles) to additionally examine gene expression of procollagen type 3, tropoelastin, lysyl oxidase, prolyl-4-hydroxylase, Smad 2, Smad 3, Smad 7, and TGF-β receptor 1. The captured bronchioles, which measured between about 75 and 200 μm internal diameter, included both epithelium and bronchiolar wall but no alveolar walls or nonbronchiolar tissues.
All bronchioles from several slides were collected on LCM caps, and each sample collection cap then placed on an Eppendorf (Mississauga, ON, Canada) 500-μl tube and stored at −80°C until the RNA extraction and isolation procedure. RNA was extracted using the PicoPure RNA Isolation kit (Arcturus) as described previously by us (4, 15). All the RNA from the small airways from a given animal was pooled as one data point. Samples typically contained approximately 50 to 100 ng of RNA.
With a dissecting microscope we then used a needle to remove any residual bronchiolar tissue that had not been picked up by the laser capture apparatus: respiratory bronchioles, the pulmonary artery branches that accompany the bronchioles (since previous studies in our laboratory have shown that these vessels express a wide variety of mediators after smoke exposure ), small pulmonary veins, cartilaginous airways, and larger arterial/venous branches. Finally, the airway/vessel-free parenchyma (Figure 1) was scraped off the slide and analyzed for expression of the same genes.
Reverse transcription and real-time PCR were performed as previously described by us (4, 15). Real-time PCR was performed on a Roche LightCycler. Each set of PCR reactions included water as a negative control, and five dilutions of standard. Standards were created by cloning part of the transcript of interest into a cloning vector (Invitrogen, Carlsbad, CA). Each insert was generated by PCR from cDNA. Known amounts of DNA were then isolated and diluted to provide standards and a regression curve of crossing points versus concentration generated with the LightCycler. GAPDH was used as a housekeeping standard and was similarly cloned. Primer sequences were as follows. Procollagen type 1 (364 bp, Genebank NM_007742): forward, GCC GAT GAT GCT AAC G; reverse, CGT ACT CGA ACG GGA AT. Procollagen type 3 (192 bp, Genebank NM_009930): forward, CAC GCA AGG CAA TGA G; reverse, GTT GGT CAC TTG CAC TG. Tropoelastin (89 bp Genebank NM_007925): forward, GAT GGT GCA CAC CTT TGT TG; reverse, CCG GTA TTT GGG TAC CAA TG. TGF-β1 (202 bp, Genebank M57902): forward, GAG GGC TTA GGG TTG G; reverse, CTT AGG ACC CCT TCC G. CTGF (360 bp, Genebank BC006783): forward, AGG AAG TAA GGG ACA CG; reverse, CTC CCC GGT TAC ACT C. PDGF-A (214 bp, Genebank M29464): forward: GCT CGA AGT CAG ATC CAC; reverse, TCT CGT AAA TGA CCG TCC. PDGF-B (330 bp, Genebank BC053430): forward, CTA CGT TCA CTT CCG GT; reverse, GCC TCG GTA TGA ATT AAA CA. Prolyl-4-hydroxylase (211 bp, Genebank BC009654): forward, CCT TGG AGA CGG TAC AT; reverse, CTA AAG GCA TCC GGC T. Lysyl oxidase (199 bp, Genebank NM_010728): forward, GAG AGG TTG GCG AAC A; reverse, AGT ACG ACT TCG GCA C. TGF-β-Receptor 1 (196 bp, Genebank NM_009370): forward, TGC TCC GTT GTA TTT GTG; reverse, AGA AAA GGT TTA GGT TAC TCA G. Smad-2 (233 bp, Genebank NM_010754): forward, AGA GAG TTG AGA CCC CA; reverse, AGA GCC TGT GTC CAT AC. Smad-3 (214 bp, Genebank NM_016769): forward, ACT GAC CTG AGT GTG C; reverse, GAT GTT TGC CTC TGC T. Smad-7 (331 bp, Genebank AF015260): forward, AGG CTC TAC TGT GTC C; reverse, ACT CGT GGT CAT TGG G. GAPDH (518 bp, from Ref. 16): forward, CGG ATT TGG CCG TAT TGG GC; reverse, TGA TGG CAT GCA CTG TGG TC.
For immunohistochemical demonstration of collagens 1 and 3, sections were pretreated with protease type XXIV (Sigma, St. Louis, MO) for 30 minutes at room temperature. Staining was overnight at 4°C using rabbit anti-collagen 1 or collagen 3 (Rockland, Gilbertsville, PA) at a dilution of 1:150. Negative control slides were incubated with nonimmune IgG instead of the primary antibody. Sections were then incubated sequentially with Dako Biotin Blocking System and biotinylated second antibody (Dako, Mississauga, ON, Canada) at a dilution of 1:1,500, followed by Vector NovaRED for color development (Vector Laboratories, Burlingame, CA). Staining of 100 randomly selected alveolar walls was assessed visually and graded from 0 to 3, and an average grade calculated for each case. For immunohistochemical staining of TGF-β1, Santa Cruz Biotechnology (Santa Cruz, CA) antibody SC-146 was used at a dilution of 1:200 followed by second antibody as above at 1:2,000. Airway grading was assessed visually as above and a mean case grade created; for the parenchyma, the number of staining cells per 25 randomly selected high power fields was counted and a mean value per case determined.
To examine apoptosis of lung parenchymal cells, staining for active caspase-3 was performed using 15-μm-thick sections (R. Tuder, personal communication) with rabbit anti-active caspase-3 (Cell Signaling Technologies, Beverly, MA) diluted 1:400 and incubated overnight at 4°C, followed by goat anti-rabbit (Dako) diluted 1:2,000, and visualized as above. Twenty-five random fields were photographed at ×250. Numbers of positive nuclei were counted in each field, and the total length of included alveolar wall was measured using ImagePro. These values were summed to give a final measure of numbers of positive nuclei per length of alveolar wall.
To examine cell proliferation, staining for MIB (Ki67) was used. Staining was performed using Dako rat anti-mouse Ki67 at a dilution of 1:200 for 1 hour at room temperature, followed by biotinyated rabbit anti-rat serum (Dako) for 30 minutes. Visualization was performed using the ABC/AP kit (Dako). Positively and negatively stained nuclei were counted in 25 fields and final results expressed as percentage of positively staining nuclei.
For the gene expression studies, groups of three animals were used for each time and treatment, and analysis of the data showed that sufficiently high levels of statistical significance were achieved with this group size. Statistical analysis of gene expression data was performed by ANOVA using SYSTAT 11 (SYSTAT Software, San Jose, CA) and comparing control to smoke-exposed animals at each time point. (For Figures E1–E21 in the online supplement, the control values for each time point have been normalized to a mean of 1.0 and the smoke values adjusted accordingly to allow intuitive understanding of how gene expression changed over time and also of differences in the relative expression levels of different genes, but the original data values were used for the statistical analysis.) In some instances, the data were log-transformed before the analysis. For constructing Figures 2 and 3, and Figures E22 and E23 in the online supplement, the mean values of control and smoke-exposed animals for each gene from Figures E1 to E21 and similar data from our previous study (4) were used to produce a mean smoke to control ratio in the airways and parenchyma at each time point. For immunohistochemical studies, five animals per treatment group were examined and data compared by ANOVA.
Figure 1 shows an example of the parenchyma remaining after removal of bronchioles and vessels; this is the tissue that was then scraped off the slide and sampled for parenchymal gene expression. An illustration of a representative laser capture microdissected small airway can be found in the online supplement to Reference 4.
Measurement of mean linear intercept indicated that there was a 35% increase in mean airspace size in the smoke-exposed animals (controls 41.0 ± 2.9 versus smoke-exposed 55.2 ± 1.0 μm, P < 0.001) at 6 months. As indicated in Reference 4, the small airways showed about a 50% increase in collagen content at 6 months.
Graphs showing the time course of gene expression for each gene newly analyzed in this study are presented in Figures E1 to E8 (airways) and E9 to E21 (parenchyma). A graphical summary of the 2-hour and 24-hour data is presented in Figures E22 and E23, and a summary of the 1-month and 6-month data in Figures 2 and 3. The graphical summaries incorporate the data on expression of procollagen type 1, TGF-β1, CTGF, and PDGF-A and -B from Reference 4 for easy comparison with the parenchymal changes; detailed original data for these five genes are available in that article (4).
In the airways at 2 hours after starting smoke exposure (Figure E22), there was an increase, often quite marked, in gene expression of procollagen type 1, TGF-β1, CTGF, PDGF-A and -B, Smad 3, and TGF-β-receptor 1; these all declined to control values by 24 hours (Figure E23) (4). Gene expression of most of the remaining genes was not increased at 2 or 24 hours, except for an increase in Smad 2 at 24 hours (Figures E22 and E23). However, after 1 month of smoke exposure (Figure 2), all genes except PDGF-A were elevated, most by considerably more than 2-fold. By 6 months of smoke exposure (Figure 3), all genes except TGF-β1 were elevated, with marked elevations (> 4-fold) of prolyl-4-hydroxylase, tropoelastin, procollagen type 1, PDGF-B, CTGF, Smad 2, Smad 3, Smad 7, and TGF-β-receptor 1, and lesser but still significant elevations of procollagen type 3, lysyl oxidase, and PDGF-A. Only TGF-β1 was not elevated at this time point.
The pattern in the parenchyma differed considerably. At 2 hours after starting smoke exposure (Figure E22), all genes except CTGF, Smad 7, and PDGF-B were elevated. In contrast to the airways, at 24 hours after a single smoke exposure, almost all the genes that were elevated at 2 hours remained elevated (Figure E23). At 1 month after starting smoke exposure (Figure 2), all of the genes examined were elevated, and some showed quite marked elevations—for example, tropoelastin expression was increased 17 times. However, after 6 months of smoke exposure (Figure 3), the pattern in the parenchyma was distinctly different from that seen previously and also different from that in the airways. Only procollagen type 1, Smad 2, Smad 7, and TGF-β-receptor 1 showed increases in gene expression, whereas prolyl-4-hydroxylase, procollagen type 3, lysyl oxidase, and TGF-β1 were significantly down-regulated, and the other genes examined were not different from control.
To determine when the parenchymal changes started to occur, we selected three genes (tropoelastin, procollagen 3, and TGF-β1) that were elevated at 1 month but were at control levels or down-regulated at 6 months, and examined gene expression at 3 months. As shown in Figure 4, by 3 months expression levels of all three genes had returned to control values. These findings suggest that loss of response necessary for repair in the parenchyma precedes the appearance of emphysema (see Discussion).
To determine whether gene expression changes correlated with protein production, we examined two important matrix proteins, collagen type 1 and collagen type 3, and also TGF-β1 by immunohistochemical staining. As shown in Figures 5A and 5D, collagen 3 immunostaining was increased in smokers at 1 month but distinctly decreased at 6 months; for collagen 1 (Figures 5B and 5D), there was a suggestion of increased staining at both time points, but this was not statistically significant. In the airways there was intense staining for TGF-β1 at both 1 and 6 months, and this was not changed by cigarette smoking (Figures 5C and 5D). In the parenchyma, staining was seen in type 2 cells and alveolar macrophages, and the number of staining cells/unit area was decreased in the smoking animals at 6 months (Figures 5C and 5D).
To investigate whether chronic smoke exposure caused a change in cell proliferation and/or apoptosis of parenchymal cells, immunohistochemical staining for MIB and active caspase-3 were performed. There was no change in cell proliferation or number of apoptotic cells between 1 and 6 months in either smokers or controls (data not shown). We consider the significance of this finding below.
It is generally accepted that proteolytic attack on the alveolar matrix mediated by smoke-evoked inflammatory cells is the driving force behind the destructive changes in emphysema (1). What has become a major focus of late is the question of why this destruction persists, and, as noted in the first part of this article, a variety of mechanisms have been postulated to explain this phenomenon.
The hypothesis of failure of lung maintenance because of apoptotic loss of lung structural cells can be approached by looking for apoptotic and proliferating cells in emphysematous lungs. Increased numbers of apoptotic cells have been reported in emphysematous compared with nonemphysematous human lungs (7, 17, 18). However, in smoke-exposed animal models, in which much milder emphysema is present, the data are more controversial; two reports (19, 20) document increases in numbers of apoptotic cells and caspase activation in the parenchyma in C57Bl/6 mice, but another study of A/J mice (21) failed to find increases after 6 months of exposure. We could not show any differences in numbers of apoptotic or proliferating cells in the current study. Thus changes in relative cell number do not appear to explain our data on relative gene expression levels.
Since destruction of matrix appears to be central to emphysema (1), and since lavage levels of the matrix (elastin) breakdown product, desmosine, correlate strongly with airspace size in a guinea pig smoking model (22), one relatively direct test of the failure to repair hypothesis is to examine changes in expression of genes related to matrix production in the parenchyma and also to compare such changes to the small airways, which become fibrotic with chronic smoke exposure in both humans and animals (acknowledging, of course, that gene expression levels and protein production levels do not necessarily correspond).
As noted in the beginning of this article, the available human microarray data on matrix production in emphysematous lungs are almost completely inconsistent from report to report (8–11), and these reports also do not separate airways from parenchyma. This separation is important because the airways and parenchyma are structurally very different (reviewed in Ref. 23) and react differently to smoke. Bronchiolar epithelium is composed of Clara cells, ciliated cells, and mucus-secreting cells, whereas in the parenchyma the epithelium is composed of type 1 and type 2 cells, and there is undoubtedly a contribution to parenchymal effects from alveolar macrophages. In addition, the airways have considerable amounts of smooth muscle in their walls. Both sites have a complement of fibroblasts, but whether these are functionally different is not known. It is possible that shifts in relative numbers of different types of cells account for change in gene expression levels over time, but it is also possible that there are functional changes in a given cell type over time.
As compared with outbred humans, who might in fact develop morphologically similar emphysema with different genetic backgrounds and possibly via different molecular pathways, inbred lines of mice offer a simpler model in which individual to individual genetic variation is minimized. Here we have separated anatomic compartments and shown that the small airways and parenchyma react to cigarette smoke exposure at a molecular level in quite different fashions, with, in a very broad sense, the airways persistently up-regulating expression of genes that control production of matrix proteins, the proteins themselves, and profibrotic growth factors and signaling molecules over time. In contrast, although the same genes were initially and rapidly up-regulated in the parenchyma, with increases discernible in as little as 2 hours after starting smoke exposure (albeit with more animal-to-animal variability in the very acute responses than is seen in the longer term), and remained up-regulated through 1 month, at 6 months expression of most of these genes had declined to control values or showed evidence of suppression. These changes in gene expression levels correlate with morphologic findings, since in both smoke-exposed guinea pigs and mice (24; J. L. Wright, unpublished observations) increases in airspace size cannot be detected at 1 month, whereas they are readily apparent at 6 months, at which time small airway remodeling, manifest as increased collagen in the airway walls, is also present (4, 22).
A brief comment about the genes that we selected for examination is in order. In terms of emphysema, procollagens type 1 and 3 and tropoelastin are matrix protein precursors that are believed to be important in providing tensile strength to, and determining compliance of, the alveolar wall (25). Our immunohistochemical evaluation confirmed that collagen 3 protein is increased at 1 month but significantly decreased by 6 months in the parenchyma, whereas collagen 1 protein is maintained (and may be increased) through 6 months, data that roughly correspond to the changes in gene expression values in the parenchyma.
Prolyl-4-hydroxylase catalyzes the formation of hydroxyproline in procollagen, and hydroxyproline is required for stability of the collagen triple helix (26). Similarly, lysyl oxidase crosslinks both immature elastin and collagen (27) to form the stable insoluble mature matrix proteins. In the present study these genes were up-regulated and stayed up-regulated in the airways, but by 6 months were actively down-regulated in the parenchyma, a process that would lead to decreased stability of crucial matrix proteins.
TGF-β1 and PDGF-A and -B are profibrotic growth factors with somewhat differing functions. TGF-β is a powerful stimulator of collagen and elastin production by fibroblasts and myofibroblasts (28), whereas PDGF primarily causes fibroblast proliferation (29), thus increasing the pool of cells upon which TGF-β can act.
TGF-β production and signaling is complex and often cell type dependent. On TGF-β binding to its receptors, there is phosphorylation of the R-Smads, Smad 2 and Smad 3, which then oligomerize with Smad 4 and are translocated to the nucleus, where the Smad complex activates or down-regulates various transcriptional programs (30). Smad 7 is an inhibitory Smad that antagonizes TGF-β signaling. Although Smad 7 is ordinarily induced by TGF-β signaling, other pathways, such as mitogen-activated protein kinase pathways, can also lead to Smad 7 production (30). CTGF is a downstream signaling molecule that is generally driven by TGF-β and is believed to be the proximate mediator of TGF-β–driven fibroblast collagen production (31).
Interpretation of our data is not straightforward. In general, increased levels of Smad 7 gene expression are associated with decreased levels of TGF-β gene expression. This appears to be true in the parenchyma, where there is not only a decrease in TGF-β1 gene expression, but also a decrease in the number of TGF-β1 immunochemically positive cells at 6 months. A lack of functional TGF-β could certainly be an important determinant of the decreases in matrix protein production, since TGF-β is required for elastin synthesis (32) and, as noted, is an important driver of collagen synthesis.
However, in the airways, there was fairly intense immunohistochemical staining for TGF-β1 in both control and smoke exposed animals at 1 and 6 mo with no obvious differences over treatment or time. Looked at one way this makes sense, since the airways become fibrotic over time and there is increased CTGF production, usually implying TGF-β signaling. But looked at another way this is hard to understand, since the increased Smad 7 gene expression and relatively decreased TGF-β1 gene expression seen in the airways at 6 months should also be associated with decreased protein production. One potential reason for these findings is that immunohistochemistry is only semiquantitative and changes from a high baseline level can be hard to detect. Further, TGF-β production is regulated at both the transcriptional and translational level so that the connection of gene expression to protein production may not be simple. There may also be increases in production of TGF-β latent binding protein in the airways, and we have documented here increases in gene expression of Smad 3 and TGF-β receptor 1, so that, overall, TGF-β signaling may be enhanced in the airways.
Why most of the genes examined are turned off in the parenchyma but not the airways is an important question that we cannot readily address in this type of study. However, cigarette smoke produces a complex inflammatory milieu that might affect matrix production on a cell type–specific basis. For example, TNF-α and IL-1β are increased in human and murine smokers (33, 34; A. Churg, unpublished data), and both these cytokines can down-regulate the expression of tropoelastin (31). In addition, murine smoke exposure models have shown that smoke elevates IFN-γ via increased production of IL-18 (35), and IFN-γ also antagonizes TGF-β signaling (36). TGF-β itself suppresses B and T cell function; thus, a lack of TGF-β may allow abnormal adaptive immune responses, and such responses have been suggested to play a role in the pathogenesis of emphysema (reviewed in Ref. 2).
In summary, we have shown for the first time in an animal model that the pattern of expression of a number of genes that control/produce a fibrotic and elastogenic milieu are different in the small airways and parenchyma and that there is indeed, in a broad sense, a failure to repair damaged matrix in the parenchyma. However, this process does not occur in a simple fashion, since early on there is a pro-fibrotic/pro-elastogenic milieu in both sites, but with longer smoke exposures these effects are turned off and the parenchyma switches to a relatively anti-fibrotic/anti-elastogenic regime, as opposed to the airways in which a pro-fibrotic regime is maintained and fibrosis develops. The data also suggest that failure of the parenchyma to repair may be related to disregulation of TGF-β signaling.
An abstract with a portion of these results has previously been published (37).
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