We found that extravascular fibrin is florid in this porcine model of SIALI and that it is distributed in the conducting airways as well as in the small airways and alveolar space. While clinical inhalation injury involves variable exposure to a complex mixture of particulates and toxic gases, this model reliably generates inhalationally-induced ALI that exhibits key features of the clinical disease. Airway casts that form within 24 h after induction of SIALI were confirmed to be fibrinous by immunohistochemical analyses. The prominent fibrinous casts in the conducting airways likely contribute to the increased airway resistance often observed in SIALI Induction of bronchospasm initiated by coagulation in the airways has previously been reported in SIALI. This may be attributable to intraluminal deposition of airway fibrin (21
). In our model, bronchoscopic removal of these casts was mandatory for survival of animals challenged with SIALI, which may have limited their detection in conducting airways of tissues harvested at necropsy. Prominent alveolar fibrin deposition likely contributes to the surfactant dysfunction, atelectasis and impaired gas exchange that characterize this model and that are known to occur in clinical SIALI.
Our data indicate that PAI-1 is induced in the lungs in SIALI, and that increased PAI-1 is temporally associated with and proximate to sites of extravascular fibrin deposition. PAI-1 was found to be induced in the epithelium and connective tissues of the airways and in the subpleural connective tissues and mesothelium within 24-48 h after injury. In addition, PAI-1 expression was increased in BAL of pigs during the progression of SIALI over this same time frame, indicating that small airway and alveolar lining fluids were enriched. By fibrin plate analyses, fibrinolytic activity was detectable in the BAL of control animals and relatively decreased in that of pigs with SIALI, indicating that induction of PAI-1 was at least in part responsible for the defect in local fibrinolytic activity in BAL. Despite evidence of relatively low levels of uPA and PL amidolytic activities in BAL, the presence of concurrently increased PAI-1 was demonstrated by reverse enzymography and likely accounts for much of the decrement of PA activity in amidolytic analyses. These results could indicate formation of “moleculer cage” type complexes between macroglobulins and uPA and PL, which we detected in PFs of animals with pleural injury (18
). Collectively, these data confirm that PAI-1 contributes to the decreased fibrinolytic activity observed in BAL of pigs with SIALI, promotes persistence of airway and alveolar fibrin deposition, and thereby contributes to abnormal gas exchange observed in this model. We found that removal of fibrinous large airway casts by bronchoscopy improved gas exchange and decreased peak airway pressure, suggesting that this approach may likewise be of lifesaving value in patients with SIALI, especially those with evidence of large airway obstruction and atelectasis.
Since parenchymal lung expression of PAI-1 was robust in SIALI, as shown by immunohistochemical analyses, we inferred that resident lung cells would substantively contribute to PAI-1 expression if challenged with components of wood bark smoke. To address this possibility, we isolated and tested the ability of WBSE to induce PAI-1 in porcine ATII, lung fibroblasts and PMCs by Western and PCR analyses. Interestingly, we found that WBSE induced PAI-1 secretion by all of these resident lung cells and that the magnitude of the response was comparable. As PAI-1 is a secreted protein, ATIIs and lung fibroblasts may contribute to the levels of PAI-1 seen in BAL after induction of SIALI, and PAI-1 is likewise induced in PMCs. While it is likely that the cells harvested from the lungs of the SIALI animals were activated, our data clearly show that they retain the ability to increase PAI-1 in response to WBSE exposure.
We next sought to test the mechanism by which WBSE induced PAI-1. It is well-known that PAI-1 gene expression is regulated at the level of transcriptional activation by mediators that like TGF-β are present in the setting of high-grade inflammation (13
). To our knowledge, however, there have been no prior studies that addressed the possibility that PAI-1 is regulated at the level of posttranscriptional control of mRNA stability in this context. We recently reported that lung epithelial cells and PMCs regulate PAI-1 at the posttranscriptional level (20
), providing us with a strong rationale to investigate whether this pathway is operative in SIALI. In that study, we reported a novel PAI-1 mRNA destabilizing interaction involving the interaction of PAI-1 mRNA with a protein we characterized as 6-PGD. TGF-β–mediated induction of PAI-1 was found to dissociate 6PGD from its specific 33 nt binding sequence within the 3′ untranslated region (3′UTR) of PAI-1 mRNA. In the present study, we report, for the first time, that a similar mechanism is operative when primary porcine PMCs, lung fibroblasts or ATIIs are stimulated with WBSE or TGF-β. Our group previously showed that PAI-1 may be induced by an alternate mechanism in which induction of PAI-1 in lung epithelial cells of mice with bleomycin-induced ALI or human ATII cells exposed to bleomycin or cigarette smoke extract occurred through interaction of p53 with PAI-1 mRNA (8
). While we found immunohistochemical evidence of induction of p53 and interstitial apoptosis of the lungs of animals with SIALI, we found no evidence of the interaction of p53 with PAI-1 mRNA by gel shift analysis. It is possible that this finding relates to differences in the interaction of the human PAI-1 mRNA binding sequence with porcine p53. Our findings are consistent with the immunohistochemical findings indicating that PAI-1 is induced in these cell types in SIALI and suggest the likely possibility that the process could contribute to PAI-1 expression in the airways, alveoli and mesothelium in pigs with SIALI.
Our observations confirm that SIALI exhibits profound impairment of the pulmonary fibrinolytic system and that PAI-1 over-expression contributes to the effect. These changes are characteristic of ALI (8
) but their contribution to the pathogenesis of ALI appears to be complex. For example, lung function is not altered in PAI-1 or fibrinogen knockout mice with ALI induced by hydrochloric acid (HCl) (22
), but the authors acknowledge that the effects of HCl on surfactant and lung dysfunction do not obviate the potentially important effects of locally increased PAI-1 and fibrin deposition in ALI on altered microvascular permeability, inflammatory cell recruitment, lung remodeling or subsequent fibrotic repair. These derangements may have special relevance to clinical SIALI, since fibrinous airway casts are a prominent feature not usually seen in other forms of ALI. It is of interest that fibrinolytic therapy has successfully been used to clear the intravascular clots and fibrinous transitional matrix, and to reverse respiratory dysfunction in various forms of ALI, in swine and in patients with ARDS (23
). To mitigate the risk of systemic bleeding, nebulized therapy with tissue plasminogen activator has more recently been used and was found to reverse pulmonary dysfunction when instituted early (within 4h) in SIALI (3
). Aerosolized anticoagulants were also shown to be beneficial in SIALI but were initiated even earlier (within 2 h of inhalation injury) in sheep (4
). These studies provide proof of principle that localized intervention that targets airway and alveolar fibrin is salutary. These studies raise the possibility that this or other relatively PAI-1 resistant agents such as single-chain urokinase (scuPA) (17
) could be of advantage when delivered by inhalation in SIALI. We found that activity of scuPA is protected from inactivation by PAI-1 in BAL fluids of pigs with SIALI (not shown), which suggests that the mechanism of protection recapitulates that which occurs in pleural fluids--where formation of scuPA or two chain uPA complexes with α-2 macroglobulin (18
In conclusion, PAI-1 is induced in the lungs of swine with wood-bark-smoke-induced acute lung injury. The increment of PAI-1 in the airways, mesothelium and alveoli is concurrently associated with decreased BAL fibrinolytic activity, and promotes extravascular fibrin in these locations. Over-expression of PAI-1 in the airways likely sustains intrabronchial fibrin casts, which are amenable to clearance by inhalational interventions that target fibrin deposition. WBSE induces PAI-1 expression in primary ATIIs, lung fibroblasts and pleural mesothelial cells in vitro. This strongly suggests that these cells may all contribute to PAI-1 expression in this model. The induction mechanism involves stabilization of PAI-1 mRNA in ATIIs and PMCs. This study represents the first demonstration of this mode of regulation of PAI-1 in this or any other form of ALI.