The role of PAI-1 in the pathogenesis of lung fibrosis was investigated using our model of fibrosis, initiated by targeted type II alveolar epithelial cell injury. We showed that an insult to the type II epithelium significantly induces PAI-1 expression in the lung. We also demonstrated that mice deficient in PAI-1 are markedly protected from epithelial injury-induced lung collagen accumulation and mortality. Furthermore, we determined that the cellular source of PAI-1 production in response to injury includes the type II AECs and two populations of lung macrophages—alveolar and exudate macrophages. In addition, we report the novel findings that PAI-1 itself mediates the accumulation of exudate macrophages and that these cells produce type I collagen. Lastly, our data revealed a strong association between the accumulation of the exudate macrophages and the development of pulmonary fibrosis in response to alveolar injury.
Prior to the initiation of these experiments, the direct relationship between PAI-1 levels and the severity of pulmonary fibrosis had been established entirely in the bleomycin model. Therefore, the possibility remained that the causal link was dependent on features unique to bleomycin injury. As such, a primary goal of this study was to determine whether PAI-1 influenced the development of pulmonary fibrosis in a separate and mechanistically distinct murine model. To accomplish this goal, we employed our recently described type II AEC injury model. With this model, we found that DT-mediated type II epithelial cell damage leads to an increase in the quantity of PAI-1 within bronchoalveolar lavage fluid. This induction of PAI-1 expression mimics the elevated levels of intrapulmonary PAI-1 observed in patients with pulmonary fibrosis of varying aetiologies [14
]. PAI-1 production is also increased in the lung following bleomycin-mediated injury [19
]. To determine whether the enhanced PAI-1 production following type II AEC injury was required for fibro-genesis, we generated transgenic mice that expressed human DTR on their type II AECs and were deficient in PAI-1. We found that PAI-1 deficiency conferred significant protection in these mice against DT-mediated pulmonary fibrosis. In fact, the lung hydroxyproline and histology in the DTR+
mice was not different from the control DTR−
group. Importantly, PAI-1 deficiency also limited the morbidity/mortality that results from the targeted type II cell insult. This decreased morbidity/mortality in the DT-treated DTR+
mice is reminiscent of that observed in PAI-1-deficient mice following both bleomycin- and hyperoxia-induced lung injury [25
]. Of note, PAI-1 inhibition was very recently reported to reduce fibrosis in yet another model of pulmonary fibrosis, intrapulmonary TGFβ over-expression [28
]. Together, these observations provide very strong evidence substantiating a critical and fundamental role for PAI-1 in the pathogenesis of pulmonary fibrosis.
Aspects of our animal model are distinct from bleomycin-mediated injury and share features of pathogenesis with the human disease which contribute to the significance of our findings. Most importantly, the DTR model recapitulates the epithelial injury that is such a prominent histopathological feature of the human disease [30
]. In fact, the generation of our DTR model was motivated by a popular hypothesis that type II AEC defects are critical for the development of lung fibrosis [32
]. This hypothesis is supported by the consistently recognized abnormalities (eg denudation and hypertrophy) in the alveolar epithelium overlying fibroblast foci, the purported lesion of active scarring. The association between mutations in type II alveolar epithelial cell-specific genes and the development of familial IPF lends additional credence to this hypothesis [33
]. Because the type II AEC injury model shares pathogenic features with IPF, we believe our data enhance the relevance of PAI-1 as a potential therapeutic target.
Our data revealed that at least two cellular sources, type II AEC and lung macrophages, contribute to the increased PAI-1 levels in the alveolar compartment of DT-injured DTR+
mice. As with our original report, we again observed persistent SPC-expressing cells in the lungs of DTR+
mice following DT administration. Importantly, these residual type II AECs contribute to the pro-fibrotic milieu through their production of PAI-1. Consistent with published literature, our present data indicate that these cells are not the only source of PAI-1 in the injured lung. In addition to type II AECs, we found that macrophages also produce PAI-1. This latter observation is consistent with data from the bleomycin model, in which microdissected type II AECs expressed ~10-fold higher levels of PAI-1, while macrophages obtained either by lavage or by microdissection expressed ~20–60-fold higher PAI-1 levels [44
]. In the present study, we further defined the production of PAI-1 by macrophage subsets and found that the accumulated non-resident exudate population most strongly expressed PAI-1. Although our results clearly implicate type II AECs and exudate macrophages as important sources of PAI-1 in the lung following a targeted epithelial injury, our results do not exclude the participation of other cells, such as fibroblasts, in the production of PAI-1.
Although many studies implicate PAI-1 as a potent fibrogeneic mediator, the elucidation of its mechanism of action in promoting collagen accumulation has been elusive. Herein, we provide results that further clarify the role PAI-1 in fibrogenesis. We report the novel finding that PAI-1 is necessary for the accumulation of exudate macrophages in the lung following type II AEC injury. These cells express collagen 1
mRNA and stain for intracellular collagen, suggesting that they contribute directly to the fibrotic process. Our data reveal that PAI-1 facilitates the accrual of exudate macrophages by mediating the recruitment of Ly6Chigh
monocytes, the precursor of exudate macrophages, into the injured lung. There exist many plausible mechanisms by which PAI-1 might influence this monocyte recruitment. For example, PAI-1 may enhance the transendothelial migration of Ly6Chigh
monocytes across the pulmonary vasculature (perhaps through its vitronectin-binding function) [42
]. Alternatively, PAI-1 may alter the expression or recognition of monocyte/macrophage chemotactic factors (ie CCL2 or CCL7). A potential effect of PAI-1 on monocyte development within the bone marrow also warrants consideration. Future studies are planned to further delineate these potential mechanisms.
In summary, we have determined that a targeted type II cell injury leads to the significant induction of PAI-1 and that PAI-1 is necessary for the subsequent fibrotic response. These findings establish a causal role for PAI-1 in a model of pulmonary fibrosis other than bleomycin, and implicate PAI-1 as a central component of the fibrogenic pathway. Our data further implicate the PAI-1 mediated accumulation of exudate macrophages as one potential mechanism whereby PAI-1 contributes to the development of lung fibrosis. Ultimately these findings further motivate the targeting of PAI-1 as a therapeutic strategy for human fibrotic disorders.