Background: Small airway remodeling (SAR) is an important cause of airflow obstruction in cigarette smokers with chronic obstructive pulmonary disease, but the pathogenesis of SAR is not understood.
Objective: To determine whether smoke causes production of profibrotic growth factors in the airway wall.
Methods: We exposed C57Bl/6 mice to cigarette smoke for up to 6 mo and examined growth factor/procollagen gene expression in laser-capture microdissected small airways by real-time reverse transcription–polymerase chain reaction.
Results: With a single smoke exposure, increases in procollagen, connective tissue growth factor (CTGF), transforming growth factor (TGF)-β1, platelet-derived growth factor (PDGF)-A and -B expression were seen 2 h after the start of smoking and declined to baseline by 24 h. With repeated exposures and at killing of animals 24 h after the last exposure, increases in procollagen, CTGF, PDGF-B, and (minimally) PDGF-A expression persisted through 1 wk, 1 mo, and 6 mo. TGF-β1 gene expression declined over time; however, increased immunochemical staining for phopho-Smad 2 was present at all time points, indicating continuing TGF-β downstream signaling. Morphometric analysis showed that the small airways in smoke-exposed mice had more collagen at 6 mo.
Conclusions: These findings suggest that smoke can induce growth factor and procollagen production in small airways in a time frame that initially is too short for a significant inflammatory response and that profibrotic growth factor and procollagen gene expression become self-sustaining with repeated smoke exposures. These results imply that the pathogenesis of and possible treatment approaches to emphysema and small airway remodeling might be quite different.
Small airway remodeling in cigarette smokers causes airflow obstruction. The pathogenesis of small airway remodeling is unknown but usually attributed to smoke-induced inflammation.
We show that small airway remodeling is caused by smoke-induced production of growth factors in the airway wall rather than inflammation, implying that growth factor antagonism may be an approach to treatment and prevention of small airway remodeling.
The pathogenesis of SAR in chronic obstructive pulmonary disease is an area of dispute. There are two general schools of thought (3). One is that SAR represents a response to repeated inflammatory insults evoked by cigarette smoke or reflects a propensity to develop abnormal inflammatory reactions to minor stimuli. In this view, the resulting changes in airway structure are a manifestation of aberrant healing induced by inflammatory cells, implying that antiinflammatory agents can be used to prevent or treat SAR. For cigarette smoke–induced SAR, this view is the predominant one in the literature (3–6); but, as pointed out in a recent review (3), there is little direct evidence to support it.
An alternative theory is that SAR is caused by excessive production of growth factors, which leads to increased muscle and fibrous tissue, and that growth factor production is a direct response to the inciting agent, possibly mediated through chronic injury or repair of the airway epithelium, but is independent of the inflammatory response (3). These findings are consistent with an increasing body of literature, largely derived from models of interstitial pulmonary fibrosis, that suggests that inflammation and fibrosis are in fact separate processes, with the latter driven by injury to and/or stimulation of the epithelium and subsequent epithelial production and release of growth factors that influence underlying mesenchymal cells (7–9).
In the present study, we have used laser-capture microdissection (LCM) of small airways from smoke-exposed mice to examine the induction of profibrotic growth factors and morphometric analysis to evaluate the development of SAR.
Some of the results of these studies have been previously reported in the form of an abstract (10).
C57Bl/6 mice were exposed either once to four 2R1 Kentucky research cigarettes (University of Kentucky, Lexington, KY), or on a daily basis (5 d/wk) to three 2R1 cigarettes for periods up to 6 mo. For the single smoke exposure groups, animals were killed at 2, 6, and 24 h; for the chronic exposure groups, animals were killed 24 h after the last smoke exposure.
After mice were killed, the lungs were removed and inflated with either cold 100% ethanol for LCM or formalin for immunochemistry and morphometry.
LCM was performed on 5-μm-thick histologic sections using an Arcturus Pixcell II (Arcturus, Mountain View, CA) LCM apparatus. All bronchioles from six histologic sections 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 (10). All the RNA from a given animal was pooled as one data point.
Reverse transcription (RT) and real-time polymerase chain reaction (PCR) were performed as described by us previously (11). Real-time PCR was performed on a Roche LightCycler (Roche, Indianapolis, IN). 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. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as a housekeeping standard and was similarly cloned. Primer sequences are listed in the online supplement
Separate sets of animals were exposed to smoke and, after being killed, their lungs were inflated with formalin at 25 cm pressure for 24 h on a continuous perfusion tank. After fixation, they were serially sectioned in a sagittal plane, and all lung sections embedded in paraffin and cut at a thickness of 5 μm.
A histologic section was stained with hemotoxylin–eosin or picrosirius red, a relatively specific stain for collagen, and total airway area per unit parenchyma as well as total airway collagen per unit parenchyma were measured using all the small airways (bronchioles) in the section. See the online supplement for details.
Immunochemistry was performed using the formalin-fixed histologic sections as described by us previously (12). For quantification of nuclear staining, color segmentation was used with the ImagePro system (Media Cybernetics, Silver Spring, MD), and the area of stained nuclei calculated and expressed per length of airway basement membrane. See the online supplement for details.
Statistical analysis was performed by analysis of variance. Details are provided in the online supplement.
Figures 1 to 3 and 5 to 6 show gene expression levels of matrix proteins and growth factors in the airway walls. For all of these figures, the 2-, 6-, and 24-h data are derived from a single exposure to cigarette smoke, with animals killed at the indicated times, and labeled “single smoke exposure” on the graphs; the 1-wk, 1-mo, and 6-mo data (labeled “chronic exposure”) are from daily smoke exposure, with animals killed 24 h after the last exposure. Group size is three animals for the acute exposure experiments and four animals for the chronic exposure experiments. In all the figures, the data for control animals are tightly clustered, a finding that indicates good RNA preservation.
Figure 1 shows type 1 procollagen gene expression in the small airways. At 2 h after smoke exposure, there was an approximately sixfold increase in gene expression and this declined over 24 h. In the chronic exposures, there was a roughly eightfold increase in gene expression at all time periods.
Figure 2 shows connective tissue growth factor (CTGF) gene expression. A pattern similar to that for type 1 procollagen was observed, with rapid decline to control levels after a single smoke exposure; however, in the chronically exposed animals, there was a progressive increase in gene expression of CTGF over time.
Figure 3 shows TGF-β1 gene expression. Again, there was a marked increase at 2 h, with rapid decline after a single smoke exposure. With chronic exposure, there was about a threefold increase in gene expression in the 1-wk animals with a progressive decline, so that at 6 mo, the smoke-exposed animals were not different from control animals.
Figure 4 shows representative immunochemical stains for phospho-Smad 2 together with a graphical representation of amount of nuclear staining per unit length of basement membrane. There was a marked increase in nuclear staining 2 h after a single smoke exposure. This declined over 24 h but still remained elevated compared with the control. With chronic exposures, positive staining was seen at all time points, albeit never at the level seen at 2 h. At all time points, the level of staining was significantly greater than control (no staining was seen in control animals at any time). Analyzing differences in staining levels over time, the 2-h time point had significantly greater staining than the 6- and 24-h time points; in the chronic exposures, there was no significant difference in staining levels over time among the three groups.
Figures 5 and 6 show gene expression of platelet-derived growth factor (PDGF)-A and -B. Both genes were markedly elevated at 2 h and declined to control levels by 24 h; for PDGF-A, no or only very small increases were seen with chronic exposure, whereas PDGF-B levels were markedly but quite variably elevated at all time points.
Figure 7 shows changes in small airway wall structure at 6 mo. We assumed that polarized picrosirius red staining indicates collagen, an assumption that is generally correct. On casual examination of routine hematoxylin and eosin stains, it was not possible to separate control from smoke-exposed airways, but increased amounts of collagen were visible on picrosirius red stains (Figure 7). There was about a 40% increase in total small airway wall area/unit parenchyma in the animals exposed to smoke, but this was not statistically different from control (p = 0.12). However, airway wall collagen as assessed by picrosirius red staining was increased by 55%, a significant difference from control (p < 0.05).
The concept of smoke-induced SAR was introduced in the late 1960s when, using airway resistance measurements, Hogg and colleagues found that distending pressures did not have an effect on lung resistance, suggesting that it was the airways rather than the emphysema that were responsible for the airflow resistance increase in emphysematous lungs (13). This was supported by data showing that pulmonary conductance correlated with mean bronchiolar diameter (14). The implication of these studies was that the small airways in cigarette smokers had functional narrowing of their lumens.
Although the physiologic effects of smoke-induced small airway remodeling are generally accepted, ideas about the pathogenesis of SAR in cigarette smokers have in many senses been derived from concepts of the pathogenesis of emphysema, especially the protease–antiprotease hypothesis. The latter states that smoke evokes an inflammatory cell influx, and that these inflammatory cells release proteases that overwhelm the antiproteolytic defenses of the lower respiratory tract, leading to proteolytic attack on the lung matrix with subsequent destruction of alveolar walls and emphysema (15).
There is very little actual information about the pathogenesis of SAR in smokers, but the fact that smoke produces an inflammatory cell infiltrate in the lungs has led to an assumption that the structural changes in the airway walls in smokers are in whole or part a direct result of inflammation (6, 16, 17). Increases in lumenal and wall macrophages, neutrophils, and CD8+ T lymphocytes are indeed a consistent finding in the small airways in cigarette smokers (4, 6, 18). Some reports have shown that there is a correlation between inflammation grade and decrements in pulmonary function (see Tables 6–9 in Reference 19 for more details), and Cosio and colleagues have proposed that it is specifically the presence of CD8+ lymphocytes in the airway wall that marks the smoker who will go on to develop structural abnormalities and airflow obstruction (17).
However, in all of these studies, other pathologic changes (airway wall fibrosis, increased muscle, squamous/goblet cell metaplasia, as well as lumenal mucus, or some subset of these parameters) also show correlations with abnormal pulmonary function. In a recent report (6), which carefully examined both inflammation and airway wall structure, the strongest associations of decreases in airflow were with airway wall thickness and lumenal mucus. Thus, there is no clear morphologic evidence that inflammation, per se, is the major determinant of pulmonary function abnormalities versus a marker of those in whom smoke produces marked airway abnormalities.
As noted previously, the idea that inflammation generally drives fibrosis in the lungs has been challenged. There is limited but intriguing evidence for the alternate theory that smoke induces profibrotic growth factors in the small airway walls, and that it is growth factor induction rather than inflammation that drives SAR in smokers. Studies on human lung tissue from cigarette smokers have consistently demonstrated the presence of TGF-β1 in small airway epithelial cells and airway wall cells, as assessed by RT-PCR to examine gene expression or immunochemistry to detect protein (20–23), and immunochemically graded TGF-β levels have been shown to correlate inversely with FEV1 (21, 22). In addition, cultured small airway epithelial cells from smokers spontaneously release TGF-β1 (22). These studies are somewhat difficult to interpret because most do not separate latent from active TGF-β, and the significance of latent TGF-β in this context is unclear. However, it is clear that TGF-β can produce SAR: Kenyon and colleagues (24) showed that administration of recombinant TGF-β1 to mice by intratracheal instillation resulted in increases in airway wall collagen, together with increased gene expression of types I and III collagen and increased total collagen content in distal airways. Of note, this process occurred in the absence of detectable inflammation.
We have previously used a rat tracheal explant system and showed that a very brief exposure of the explants to smoke in vitro resulted in the rapid up-regulation of CTGF, TGF-β1, and procollagen type I gene expression, together with immunohistochemical evidence of TGF-β signaling in the form of phosphorylated Smad 2 in the epithelial and subepithelial nuclei within 1 h (12). In a cell-free system, smoke-conditioned medium oxidized recombinant TGF-β latency-associated peptide, and activated latent TGF-β1 via an oxidant mechanism (12). The explants are free of smoke-evoked inflammatory cells, and thus our results support the idea that smoke can directly induce growth factors in the airway walls through an oxidant mechanism that is independent of inflammation.
In the current study, we have begun to investigate the issue of profibrotic growth factor production in the small airways in vivo. Our data show clearly that there is a rapid and marked up-regulation of growth factor production as well as production of type 1 procollagen very shortly after the beginning of smoke exposure, and that all of these parameters decline to control levels by 24 h after a single smoke exposure. This decline is in sharp contrast to the smoke-induced inflammatory response that increases over 24 h (and is not statistically increased until 24 h) under similar experimental conditions (25); thus, if inflammation was driving production of growth factors, we would expect the reverse of the pattern we observed. These findings suggest that at least the initial response to cigarette smoke is unrelated to evoked inflammation and probably reflects direct induction of growth factors by smoke, although we cannot rule out the possibility that inflammatory cells intrinsic to the airway wall—for example, resident tissue macrophages—might be playing a role.
With repeated exposures, there is continuing production of growth factors and increased type 1 collagen production, but with the difference that increases in gene expression no longer return to control values by 24 h; rather, the process appears to become self-sustaining. It is possible that this effect is driven by smoke-evoked inflammation, because neutrophils and macrophages release oxidants when stimulated, and in fact, inflammatory cells from smokers show enhanced levels of oxidant release (reviewed in Reference 26). There is also increasing evidence that neutrophils can mediate active TGF-β release via nonoxidant mechanims, because neutrophil elastase can remove the TGF-β small latent complex from the TGF-β large latent complex, and neutrophil-derived matrix metalloproteinase (MMP)-9 can then remove the latency-associated peptide, freeing active TGF-β (27). Alternately, repeated exposures to smoke may continue to cause oxidant-mediated release of TGF-β driven by oxidants in the smoke itself. Further experiments will be needed to determine which of these ideas is correct. Regardless of mechanism, our results emphasize the idea that profibrotic growth factors drive small airway remodeling.
TGF-β signals through phosphorylation of cytoplasmic receptors called Smads by TGF-β receptor 1 kinase. Once phosphorylated, Smad 2 and 3 translocate to the nucleus, where, with other cofactors, they activate a variety of genes that mediate TGF-β–driven effects. One of these genes is CTGF, which is believed to be the proximate mediator of collagen induction in fibroblasts. The present results show that there are persistent increases in nuclear phospho-Smad 2 as well as increases in gene expression of CTGF throughout 6 mo of smoke exposure, both results implying that there is continuing TGF-β signaling; moreover, as pointed out by Kelly and colleagues (28), other profibrotic growth factors generally do not induce CTGF. The presence of continuously elevated CTGF levels appears to be of particular importance; Bonniaud and colleagues (29) have suggested that CTGF is necessary for the progression and persistence of a fibrogenic response, possibly because the combination of CTGF and TGF-β activity induces tissue inhibitors of metalloproteases (TIMPs), which prevent degradation of matrix.
In a recent commentary, Chua and coworkers (30) suggest that TGF-β is the preeminent fibrogenic mediator in terms of producing interstitial fibrosis in the lung, as in fact it appears to be in pulmonary fibrosis in general (7). Animal experiments have shown that TGF-β1, and more specifically epithelial expression of TGFβ1, drives airway remodeling in murine models of asthma (28, 31). Our current data suggest that this is likely true of small airway remodeling induced by cigarette smoke.
Given the evidence just discussed that there are persisting increases in TGF-β signaling over time in the small airways in our model, the apparent down-regulation of TGF-β1 expression with long-term exposure is surprising, particularly since there is good evidence (elevated phospho-Smad-2 and CTGF levels) of continuing TGF-β downstream signaling. TGF-β1 can act via an autocrine pathway involving Ras and mitogen-activated protein (MAP) kinases to activate AP-1 and induce its own gene expression (32), and we have observed this phenomenon in rat tracheal explants exposed to smoke (12). Depending on cell type, the Ras/MAP kinase pathway can also activate Smads; for example, in hepatic stellate cells, c-Jun-N-terminal kinase (JNK) can directly phosphoryate Smad 2 and 3 (33). This phenomenon might explain the persisting Smad-2 phosphorylation and CTGF production that we demonstrate in the face of decreasing TGF-β1 gene expression. Another less likely possibility is that CTGF production is driven independently of TGF-β production; for example, thrombin has been shown to cause fibroblasts to produce CTGF (34) and cigarette smoke causes increased capillary permeability, so serum coagulation proteins can enter the airspaces. However, this would not explain the increased phospho-Smad-2 signals. It is also important to bear in mind that production of active TGF-β is not a straightforward gene-to-protein process, but rather goes through several different preformed latent complex steps, and alterations in the amount of latent protein, which we cannot measure, might well be occurring, independent of TGF-β gene expression.
Although TGF-β is a potent inducer of collagen production by fibroblasts and myofibroblasts, the major effect of PDGF is as a mitogen for these cells, although PDGF can also enhance collagen production (35). PDGF has been shown to be produced by airway and alveolar epithelial cells in fibrotic conditions, and inhibition of PDGF signaling prevents fibrosis in some murine models (36–38). Of note, by itself, adenoviral overexpression of PDGF-B in the lung produced only a mild fibrotic response; however, when combined with TGF-β1, there was a marked increase in mesenchymal cell proliferation and deposition of collagen (39). It has also been suggested that PDGF may play a role in airway remodeling in asthma (40, 41). Although we do not know the mechanism behind PDGF production in our model, it has been reported that TGF-β can induce expression of PDGF in blood monocytes (42) and in fibroblasts in patients with scleroderma (35), and a similar process may be occurring here. Increased immunochemically detectable amounts of PDGF have been seen in airways from cigarette smokers (43).
Our current data thus suggest that smoke induces a particularly potent set of profibrotic cytokines that have the potential for long-term fibrogenesis in the walls of small airways, and that such fibrogenesis does in fact occur. As is true of most rodent models of smoke exposure, morphologic changes tend to be subtle, and in fact, casual observation of hematoxylin-and-eosin–stained sections does not immediately indicate differences between smoke-exposed and control animals. However, staining with picrosirius red, which is reasonably specific for collagen, and careful morphometric analysis indicated that there is a long-term increase in collagen in the small airway walls—that is, our model system does develop SAR.
To our knowledge, this is the first clear demonstration that smoking induces profibrotic growth factor production in the walls of small airways. These findings need to be interpreted cautiously, because gene expression changes are not always translated into protein changes, or the translation is complicated, as is true for TGF-β. On the other hand, the presence of increased phospho-Smad-2 staining and increased CTGF expression indicate that there is increased active TGF-β protein release. Nonetheless, our results are of potential importance to the treatment and prevention of SAR, because they imply that the pathogenesis of SAR and emphysema may be quite different and may require different therapeutic/preventive interventions; for example, inhibitors of TGF-β signaling might be useful in SAR, but inhibitors of proteases required in emphysema. Further experiments are needed to evaluate these ideas.
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