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* From the Division of Pulmonary and Critical Care Medicine, University of Michigan Health System, 6301 MSRB III, Box 0642, 1150 W Medical Center Dr, Ann Arbor, MI 48109-0642
A fibroproliferative response begins almost immediately after the onset of acute lung injury in an attempt to repair the damage done to the alveolocapillary wall. Inflammatory cell accumulation and the entry of plasma into the airspaces alters the alveolar microenvironment. Acquired alterations in parenchymal/stromal cell phenotypes likely create tissue environments that either steer the progression of tissue remodeling toward aggressive fibrosis or toward the restoration of normal alveolar architecture. The molecular mechanisms involved in the healing process must be understood if clinicians are to counteract progressive fibrotic responses.
Acute Lung Injury
ARDS is characterized by widespread and severe damage to the epithelium and endothelium of the alveolus. Although the causes of ARDS are diverse, the sequence of morphologic features in this syndrome is similar irrespective of the cause. The alveolar injury is diffuse and temporally uniform likely because of more or less synchronous injury to large areas of the alveolar surface. Endothelial cells, which line the microcirculation, are severely injured. The extended cytoplasm of type I epithelial cells is likewise injured and these cells detach from the basement membrane. With destruction of both the endothelial and epithelial sides of the alveolar-capillary barrier, fluid and cells extravasate from the vasculature into the interstitium and the alveolar spaces. Both neutrophils and mononuclear cells are recruited into the interstitium and the airspaces of the lung. Hyaline membranes, composed of cell debris, fibrin, and other plasma proteins, form early in the course of this injury, principally along alveolar ducts.1
Repair Mechanisms
The repair response to this injury is evident within a few hours. Alveolar exudates containing fibrin fragments, fibronectin, and other matrix components provide a three-dimensional scaffold that maintains the alveolar architecture and prevents the immediate adhesion of exposed sticky basement membranes. This three-dimensional matrix also provides the scaffold for cell migration of inflammatory, epithelial, mesenchymal, and endothelial cells. Effective repair of the air lung interface and the interstitium involves processes that do the following: (1) promote the removal of intra-alveolar debris; (2) restore the extracellular matrix through tightly regulated fibroblast recruitment, proliferation, and differentiation; (3) promote reepithelization of the alveolar surface; and (4) regulate the formation of new capillaries (angiogenesis). The presence of an intact basement membrane is likely crucial to this process. A preserved basement membrane provides recruited and proliferating cells with migratory pathways that ensure the preservation of normal pulmonary architecture. A preserved basement membrane provides the framework on which tissue remodeling and the restoration of normal architecture occurs.2 3 If repair of the alveolar surface wound is dysfunctional, cells, capillaries, and connective tissue matrix may fill airspaces that must remain free of these tissues for effective gas exchange to occur.4
A fibroproliferative response and excessive accumulation of lung extracellular matrix are frequently noted in patients with ARDS. Histologic evidence of pulmonary fibrosis has been noted as early as 36 h after the onset of ARDS; increased total lung collagen is present in more established ARDS (12 to 28 days).5 6 Most patients dying of ARDS have morphologic evidence of pulmonary fibrosis. More recently, biopsy evidence of pulmonary fibrosis has been closely related to fatality in established ARDS.7 Additionally, increased levels of type III procollagen in BAL fluid has been strongly associated with increased risk for fatal outcome.8 In aggregate, these studies suggest that therapeutic interventions to influence mechanisms involved in the fibroproliferative response would have a beneficial effect on survival in ARDS. Although our understanding of the cellular and the molecular basis for the fibroproliferative response is still rudimentary, new findings regarding signals that influence the fibroproliferative response offer tantalizing, novel therapeutic options (Fig 1) .
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During acute lung injury, the alveolar spaces are filled with injured epithelial cells, inflammatory cells, edema, and accumulations of fibrin-rich airspace exudates (hyaline membranes). This exudative phase of acute lung injury represents a rapid, marked change from circumstances present in the normal alveolus. The normal alveolar space has net fibrinolytic activity due to the presence of urokinase-type plasminogen activator (uPA).9 10 Accordingly, the normal alveolar space efficiently clears intra-alveolar fibrin. Fibrin accumulates within the alveolar space of patients with ARDS, in part, because the fibrinolytic activity of BAL fluid from patients with ARDS is suppressed. The impairment in fibrinolysis is due to a decrease in urokinase protein and an increase in the inhibitors of both urokinase and plasmin.9 Changes in alveolar epithelial cells may account for a significant portion of the decrease in urokinase activity. Monolayers of normal alveolar epithelial cells can lyse a plasma-derived fibrin matrix that is formed over their surface.11 They do so by expressing urokinase that activates the plasminogen present within the plasma clot. Receptors for urokinase, which focus fibrinolytic activity, are present on the surface of epithelial cells. Epithelial cells are known to influence the fibrinolytic balance within the airspace by synthesizing both urokinase and plasminogen activator inhibitor.12 Accordingly, the persistence of fibrin in fibrotic lung disease may be due, in part, to loss of epithelial cells or to alteration in their fibrinolytic function.
An influx of coagulation factors into the alveolar space across a damaged alveolar capillary membrane also contributes to the accumulation of intra-alveolar fibrin. Normal alveolar surfaces are rich in tissue factor/factor VII and urokinase but are deficient in the distal substrates of both coagulation cascades. During acute lung injury, alveolar procoagulant activity increases in a fashion that correlates temporally with fibrin deposition. An influx of plasmin inhibitors of uPA and plasmin may be important contributors to the antifibrinolytic state of the alveolus during early phases of ARDS.13 14 15 Thus, increased permeability of the alveolo-capillary membrane also plays a crucial role in fibrin deposition by providing distal clotting factors to an active procoagulant environment while also providing antifibrinolytic substances from the plasma.
The removal of intra-alveolar fibrin is crucial to the resolution of acute lung injury. If extravascular fibrin is cleared in an orderly fashion, reconstitution of a normal alveolar space is possible. If fibrin remains, fibroblasts migrate into the fibrin matrix and secrete interstitial collagens. Fibrotic scars will form as intra-alveolar buds, thickened alveolar walls, or obliterated alveolar spaces depending on the location and extent of the residual exudate.4
Alterations in the fibrinolytic environment of the alveolar space during inflammatory injury have been shown to influence the subsequent development of pulmonary fibrosis.16 The lungs of transgenic mice overexpressing plasminogen activator inhibitor (PAI-1) contain significantly more hydroxyproline than littermate control mice following administration of bleomycin. The lung hydroxyproline content of bleomycin-treated mice completely deficient in PAI-1 was not significantly different than that of control animals receiving saline solution. Thus, PAI-1 overexpressing mice experience greater fibrosis than wild-type mice, while mice homozygous deficient for PAI-1 were protected from fibrosis.
These findings suggest that therapeutic interventions designed to enhance fibrinolysis such as administration of either plasminogen activators or inhibitors of PAI-1 might limit the development of pulmonary fibrosis that occurs in ARDS. Fibrolytic activity within the alveolar space can be manipulated with adenoviruses containing uPA complementary DNAs. uPA-containing adenoviral vectors substantially increased uPA activity in lung cells in vitro and in murine lungs in vivo. A single endotracheal instillation of adenovirus containing uPA substantially increased uPA activity within BAL fluid for at least 2 weeks. Zymographic analysis showed that the increased activity was caused by enzymes of the molecular size appropriate for the uPA transgene products. The adenovirally mediated increase in uPA production predominated over the PAI-1 known to be produced by both alveolar epithelial cells within murine lungs. Importantly, the increased uPA activity induced by gene transfer was sufficient to accelerate the lysis of fibrin-rich matrices formed from plasma. Plasma-derived fibrin rich matrices overlaid on A549 cells infected with these uPA vectors were lysed efficiently in a dose-dependent fashion. Similarly, fibrin matrices formed within intact lungs that had been infected with these uPA-containing adenoviruses were also lysed more rapidly when compared with matrices from noninfected or control virus-infected lungs. The therapeutic potential of uPA gene transfer in fibrotic lung disease is being investigated.17
Restoration of Extracellular Matrix
Repair of the interstitium requires the recruitment of fibroblasts and the tightly regulated replication of fibroblasts. These mesenchymal cells restore the extracellular matrix by the orderly deposition of connective tissues.
Growth factors capable of promoting mesenchymal cell migration and proliferation are present on the surface of the airspace in patients with ARDS. Three peptides related to platelet-derived growth factor have been identified and characterized in lavage fluid obtained from ARDS patients.18 The cellular sources of the peptides is uncertain; platelets, mononuclear phagocytes, endothelial cells, and autocrine production by fibroblasts are possibilities. Other growth factors likely participate in the fibroproliferative response. Epidermal growth factor and an insulin-like growth factor have been identified in the BAL fluid of patients with acute lung injury.
While exogenous growth factors likely play a crucial role in regulating fibroblast behavior following acute lung injury, mesenchymal cells from injured lungs are capable of sustained proliferation in the absence of exogenous peptide growth factors.19 This altered proliferative phenotype is stable for at least five subcultivations and does not depend on autocrine release of trophic factors.
Prostaglandin E2 (PGE2) is an extensively studied downregulator of fibroblast function. PGE2 has been shown to decrease fibroblast proliferation and reduce collagen levels by inhibiting its synthesis and promoting its degradation.20 21 22 Since PGE2 is a major eicosanoid product of fibroblasts, it is plausible that this molecule might regulate fibroblast function in an autocrine fashion.
Lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis have a diminished capacity to synthesize PGE2 and to express cyclooxygenase-2 (COX-2).23 Fibrotic lung fibroblasts produced 6.5-fold less immunoreactive PGE2 per microgram of protein than did normal fibroblasts. Fibrotic lung fibroblasts exhibited diminished capacity to synthesize PGE2 following ionophore stimulation as well. The defect in PGE2 synthesis exists at the level of the COX-2 enzyme itself. Unlike normal fibroblasts, fibrotic lung fibroblasts were incapable of increasing COX-2 activity after pretreatment with the agonists lipopolysaccharide (LPS), phorbol myristate acetate, and interleukin-1ß. The inability to augment COX-2 metabolic activity correlated with a failure to increase steady-state levels of COX-2 protein and messenger RNA. A defect in this homeostatic process might promote or sustain the fibroproliferative process in the lung. In the absence of COX-2 induction, fibroblasts would be unable to increase their PGE2 synthetic capacity and accordingly, these fibroblasts would be unable to limit cellular proliferation, collagen synthesis, and production of inflammatory mediators. Although only a small number of patients have been studied, a significant inverse correlation has been observed between the PGE2 synthetic capacity of fibroblasts obtained from patients with idiopathic pulmonary fibrosis and fibrosis scores of lung tissues from which these fibroblasts were obtained.
These observations may have therapeutic ramifications. Augmentation of PGE2 levels in the lung via aerosolization of stable PGE2 analog or via transfection of the complementary DNA encoding COX-2 has the potential to reduce fibrosis and ultimately to improve the prognosis for patients with fibroproliferative lung disease.
Numerous phenotypic differences have been noted between primary fibroblast lines isolated from inflamed tissues and primary lines established from control adult lung tissue. Phenotypic differences have included altered proliferative potential, matrix gene expression, cell surface marker expression, and arachidonic acid metabolism. Thus, in the context of inflammatory and/or fibrotic lung disease, fibroblasts are converted to a new, differentiated state. The possibility exists that cells thus converted may be able to perpetuate the fibroproliferative response even in the absence of external signals. Both the acquisition of processes that upregulate fibroblast function and/or the loss of factors that downregulate fibroblast function could be important in this paradigm.
Reepithelization
Denuded alveolar basement membranes must be resurfaced with a continuous epithelial cell lining during successful repair of inflammatory lung injuries. The type of epithelial cell that repopulates the alveolar space following acute lung injury likely depends, in part, on the extent of injury. Type II cells proliferate and differentiate into type I cells in minimally damaged areas of lung.24 Bronchiolar epithelial-like cells likely repopulate extensively damaged areas where no type II epithelial cells survive.25 Type II epithelial cells are the predominant cell type that covers alveolar surfaces following injuries that are intermediate between these two extremes.
Alveolar repair is dependent on both proliferation and differentiation
of type II cells. The mechanisms involved in reepithelization in
vivo are not known but are likely mediated by various growth
factors and their receptors that direct proliferation and
differentiation of type II epithelial cells. Keratinocyte growth factor
(KGF) and hepatocyte growth factor are mitogenic for type II
cells.26
KGF has been shown to be a very potent mitogen
for rat type II cells in vivo.27
Granulocyte-macrophage colony-stimulating factor (GM-CSF) may also play
a role in type II cell proliferation. Transgenic mice in which the
human surfactant protein-C gene promoter was used to direct expression
of mouse GM-CSF evidenced progressive pulmonary type II cell
hyperplasia.28
Type II epithelial cells express the GM-CSF
receptor 
-subunit providing evidence that GM-CSF can act directly on
type II cells. GM-CSF stimulates bromodeoxyuridine uptake in type II
epithelial cells in vitro supporting the concept that GM-CSF
may play a role in type II cell proliferation in vivo. While
GM-CSF increased DNA synthesis, GM-CSF alone was not sufficient to
increase proliferation in type II cells in culture. GM-CSF may act
directly on type II cells to regulate proliferation, differentiation,
or apoptosis, or to mediate other autocrine pathways influencing type
II cell proliferation or differentiation (Fig 2)
.
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Conclusion
Effector molecules likely influence the fibrotic process via both autocrine and paracrine loops. Processes important in effective repair of the air lung interface include the removal of intra-alveolar debris, the restoration of the extracellular matrix, the reepithelization of the alveolar surface, and the tightly regulated formation of new capillaries. Altered secretory phenotypes in each of these processes have been identified and represent potential targets for future therapeutic interventions.
References
This article has been cited by other articles:
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  E. Wang, M. Simard, N. Ouellet, Y. Bergeron, D. Beauchamp, and M. G. Bergeron Pathogenesis of Pneumococcal Pneumonia in Cyclophosphamide-Induced Leukopenia in Mice Infect. Immun., August 1, 2002; 70(8): 4226 - 4238. [Abstract] [Full Text] [PDF]
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E. Wang, N. Ouellet, M. Simard, I. Fillion, Y. Bergeron, D. Beauchamp, and M. G. Bergeron Pulmonary and Systemic Host Response to Streptococcus pneumoniae and Klebsiella pneumoniae Bacteremia in Normal and Immunosuppressed Mice Infect. Immun., September 1, 2001; 69(9): 5294 - 5304. [Abstract] [Full Text] [PDF]
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