Abstract
Substantial challenges remain in our understanding of fibrotic lung diseases. Nowhere is this more true than in the elucidation and verification of the pathogenetic basis upon which they develop. Scientific progress, most recently in the field of experimental therapy, has relied closely on interpreting data derived from animal modeling. Such models are used to identify the cellular interactions and molecular pathways involved in lung tissue repair and fibrosis. Over the coming years, the significance of new discoveries will continue to be evaluated using the in vivo analysis of animal models substituting for patients with actual pulmonary fibrosis. The commonest strategy to induce experimental pulmonary fibrosis is by directly administering a profibrotic agent to either wild-type animals or those that bear a specific genetic modification. The creation of new models has been greatly enhanced by the availability of stem cell lines and methods for introducing genetic mutations into these cells. Despite an increasing choice of models, there are still good reasons to continue adapting and using one of its earliest examples, the bleomycin model, in post-genomic pulmonary fibrosis research. A brief review of the exacting requirements of such research will place the strengths of this particular model in perspective.
In preparing his text of human anatomy, De Humani Corporis Fabrica, Andreas Vesalius (1514–1564) used living sows to investigate the neurological control of phonation. Claude Bernard (1813–1878), the father of modern physiology, also based his seminal work Experimental Medicine on interpreting observations made in mammalian models (1). Over the past century, the growing choice of animal models has helped drive the demand for more consistent and accurate representation of human disease in vivo. The reliance on stringently produced and biologically robust animal models is set to expand further as translational urgency of the Human Genome Project becomes a key priority. One area of pulmonary medicine that is heavily dependent on disease modeling in animals is that of pulmonary fibrosis.
Idiopathic pulmonary fibrosis (IPF) is the commonest form of the interstitial pneumonias in man (2). The lack of a true experimental analog of IPF is a key factor hampering research into this important disease. Unlike IPF, however, other fibrotic lung diseases have identifiable etiologies, including pulmonary fibrosis that complicates previous tuberculosis, connective tissue disorders, or exposure to inorganic fibers related to occupational hazards (3). Interestingly, the inhalation of crystalline particles of asbestos and silica appears to induce quite similar pulmonary changes in humans and rodents (4). In any case, the many pathologic similarities between the fibrotic reaction in human and animal lungs continue to form the basis upon which the use of animal models is so entrenched in the study of pulmonary fibrosis.
The use of animals to replicate fibrotic lung disease produces “on demand” pulmonary fibrosis. Conventional methods of inducing experimental pulmonary fibrotic reactions include the direct pulmonary instillation of a fibrogenic agent or exposure of a susceptible host to thoracic irradiation (5). While all the common approaches can generate varying degrees of parenchymal lung scarring, no single method has yet been able to mimic every aspect of human pulmonary fibrosis. In the majority of cases, the final choice of animal model is dictated by a knowledge of the unique features of the fibrotic disorder under study, the scientific question posed and logistical considerations of experimentation.
Over the last 30 years, scientific research has dramatically increased understanding of the basic pathology of the pulmonary fibrotic response. Although this may be attributed in large part to advances in molecular techniques, it is not accidental that this period also coincides with major advances in transgenic technology. Early studies demonstrated that C57/BL6 mice were consistently prone to bleomycin-induced pulmonary fibrosis, whereas Balb/c mice were inherently resistant (6). Transgenic approaches have since produced animals with genetic defects that influence fibrotic susceptibility, including the abnormal expression of certain cytokines (7), fibrinolytic pathway proteins (8), extracellular matrix components (9, 10), and tissue proteinases (11). Animal models are often most useful at revealing disease susceptibility factors or relevant biomarkers pertinent to the human situation when interpreted in the context of known clinical or pathologic attributes of the latter. However, although the nature of the inciting lung injury in many forms of human pulmonary fibrosis is poorly understood, the progression from experimentally induced lung damage to fibrotic remodeling is more linear and predictable in animals. Hence, the ease by which pulmonary fibrosis is produced in a particular model and how closely it mimics a recognized clinical entity will continue to determine the relevance of each animal model to the study of fibrotic lung disease.
Pulmonary fibrosis encapsulates the final pathologic outcome of excessive extracellular matrix deposition in the lung parenchyma. Although most animal models can replicate the major structural and biochemical abnormalities of the disease, the natural history of the fibrotic response can differ significantly between humans and animals. Cautious interpretation is crucial when data from such models are extrapolated to the clinical setting. For one thing, the insidious progression of human pulmonary fibrosis is almost impossible to reproduce accurately. In addition, the factors that regulate or promote the transformation of uncomplicated lung repair in humans to self-perpetuating pulmonary fibrosis remain incompletely understood (12). Furthermore, cessation or removal of the inciting agent in some animal models may allow the previously induced fibrotic changes to regress, a concept that is almost alien to human lung fibrosis. Taken together, these observations indicate that experimentally produced fibrotic changes act in lieu of, but do not equate to, a disease state.
A complex series of tissue reactions have to take place before fibrous connective tissue accumulation can occur (Figure 1)
Figure 1. Changes in multiple pathways link the progression of lung injury to the excessive accumulation of extracellular matrix that characterizes pulmonary fibrosis.
[More] [Minimize]Recent studies employing high throughput genomic profiling have characterized some of the gene changes that occur during the development of pulmonary fibrosis. Microarray analysis has shown that the instillation of bleomycin, a profibrotic drug, alters the gene transcription pattern in the mouse lung by increasing the expression of proinflammatory mediators, certain components of the pulmonary extracellular matrix, and genes that are induced by transforming growth factor-β (TGF-β) (14). This high-resolution technique provides a sensitive method for assessing coordinate gene expression programs, including subtle changes in apparently unrelated genes. Detailed genetic profiling in the murine lung may help identify the genes that regulate disease susceptibility or progression in human pulmonary fibrosis.
A major limitation of all experimental models of pulmonary fibrosis is their dependence on single snapshot views of what is a chronic and progressive disease process. Pulmonary fibrosis in man results from months, if not years of aberrant lung matrix remodeling. Animal models attempt to reproduce this sequence over much shorter periods of time, usually in response to a specific episode of lung injury. Hence, their use is based on two assumptions: one, that the induced lung damage will mature stepwise into progressive interstitial and alveolar fibrosis, and two, that the pathologic features revealed at the time of analysis somehow mirror a particular stage of the modeled human disease. In short, the unpredictability of fibrotic disease evolution and its tendency to intensify over time become secondary considerations. Too often, animal-to-human species variability too is forgotten as the morphologic characteristics in these models become viewed as analogous to those in fibrotic human lungs.
By what yardstick should pulmonary fibrosis be assessed? The extent of fibrotic lung disease is determined using composite information derived from radiologic imaging and physiologic testing. However, such modalities are neither feasible nor of proven value in experimental pulmonary fibrosis. Consequently, the most widely used parameters of pulmonary fibrosis in animal models remain the quantitative measurement of lung collagen content and histologic analysis of extracellular matrix deposition (15). Of course, some animal models induce these changes more consistently than others. Although the approach of animal modeling may appear reductionist by focusing on isolated molecules or signaling pathways, it remains true that only in vivo models have the potential to recapitulate the complex genetic, biochemical, and environmental interactions that combine to produce pulmonary fibrosis. In contrast, in vitro systems are limited to probing particular cellular or molecular responses that in isolation are too remote from actual lung pathophysiology.
Current concepts of lung fibrosis acknowledge that concomitant or antecedent tissue injury may be a key factor in the fibrogenic process. Up until now, research in pulmonary fibrosis has relied on animals with either single gene defects or single mediator abnormalities. By highlighting cellular behavior or biochemical pathways in isolation, such an approach runs the risk of oversimplifying or biasing the interpretation of tissue pathology. In recent years, genetic engineering has produced animals that develop spontaneous age-related lung fibrosis, although these examples remain uncommon (16). Nonetheless, the reactive phenotypes (i.e., those that develop pulmonary fibrosis following lung injury or gene activation) still appear to hold the most information for researchers. The incorporation of an exogenous lung injury stimulus to initiate aberrant lung matrix production in animals with targeted gene defects likely increases the predictive value of such models.
A number of exogenously administered agents can induce pulmonary fibrosis in a variety of animal species (Table 1)
Exogenous Agent/Approach | Nature of Tissue Damage | Animal Species Used |
|---|---|---|
| Bleomycin | Oxidant-mediated DNA scission leading to fibrogenic cytokine release | Mice, rats, hamsters, rabbits, dogs, primates, pheasants |
| Inorganic particles (silica, asbestos) | Type IV hypersensitivity reactions with or without granuloma formation | Mice, rats, hamsters, sheep, rabbits |
| Irradiation | Free radical-mediated DNA damage | Mice, rats, rabbits, dogs, hamsters, sheep, primates |
| Gene transfer (TGF-β, IL-1β, GM-CSF) | Downstream activation of specific cytokine pathway/s | Mice, rats |
| Fluorescein isothiocyanate | Incompletely understood. Presumed T-cell-independent. | Mice |
| Vanadium pentoxide | Incompletely understood. An inorganic metal oxide. | Mice, rats |
| Haptenic antigens (e.g. trinitrobenzene
sulphonic acid compounds) | Recall cell-mediated immune response | Mice, hamsters |
Because pulmonary fibrosis is characterized by increased cellularity and quantities of soluble inflammatory mediators, many early studies of experimental pulmonary fibrosis focused on changes in inflammatory and immunoregulatory phenomena. Many of these have yielded conflicting findings; consequently, the role that chronic inflammation plays in driving the fibrotic response has remained controversial (17, 18). The notion that inflammation may simply represent an important but dispensable event in pulmonary fibrosis has been proposed based on clinical and histopathologic observations in human lung tissue (19). In addition, there are also animal models in which pulmonary fibrosis coincides with or only develops after the cessation of a preceding inflammatory reaction (9, 20). Although each side of the argument may be equally compelling, a clearer picture is certain to form in the future as more detailed observations from well-conducted animal studies become available.
The use of genetically engineered mice to assess the role of specific genes or proteins in the development of pulmonary fibrosis has expanded significantly in recent years. Genetic manipulations of germ cells and the crossbreeding between two mutants are readily feasible in animals. Selective inbreeding followed by eventual exposure to an external fibrogenic stimulus further broadens the utility of these models. However, such animals may have a permanent or complex phenotype that might complicate the accurate assessment of specific molecular interactions in the fibrosing lung.
Already, the value of lung-specific expression of genes whose products alter the tissue repair potential in the alveolar microenvironment has been recognized. Adenoviral-mediated delivery of granulocyte macrophage–colony-stimulating factor, tumor necrosis factor-α, and interleukin-1β all produce variable degrees of pulmonary fibrosis that share morphologic similarities with that induced by pulmonary TGF-β overexpression (21). Conditional overexpression of “knocked-in” human genes in murine lungs have provided an additional resource with some unexpected findings. While transgene overexpression of endothelin-1 induces progressive pulmonary fibrosis, overexpression of platelet-derived growth factor-B only produces focal fibrotic lesions combined with features of alveolar emphysema (22, 23). Contrary to expectation, transgene overexpression of insulin-like growth factor-IA does not promote lung fibrogenesis, even though a role for IGF-IA in this regard has been implicated in previous studies (24). Nevertheless, such models are valuable because they permit pulmonary expression of a gene of interest at a time when lung development and maturation have been completed. Although the nature of the lung injury in many human forms of pulmonary fibrosis is poorly understood, prior alveolar damage is still thought to be pivotal to the development of subsequent fibrosis. Herein lies the second caveat. As long as the view that lung damage is a precursor of pulmonary fibrosis prevails, the induction of appropriate lung injury preceding pulmonary fibrosis will remain an integral requirement of any animal model of this disease. However, it is not known whether the chronic progression of fibrosis is as dependent on prior lung injury.
One focus of pulmonary fibrosis research is the accumulation of evidence from animal studies that TGF-β is the pre-eminent fibrogenic mediator in the lung. In the embryonic period, the absence of TGF-β is associated with perinatal lethality (25). Various rodent models have shown that enhancing the generation of active TGF-β at sites of repairing lung matrix promotes the formation of pulmonary fibrosis (9, 26, 27). Thus, gross amplification of its tissue repair properties beyond the perinatal period in response to lung injury allows this particular mediator to dominate fibrotic lung remodeling.
One of the earliest and still the most widely used animal model of pulmonary fibrosis is that of bleomycin instillation. This approach involves the parenteral administration of a drug with a predilection for causing dose-dependent fibrotic lung abnormalities in both humans and animals. While it relies on the induction of a “known” event of lung injury to initiate the fibrotic response, the recognition that it replicates many histologic features of human pulmonary fibrosis has contributed to its continued popularity.
Over the last three decades, the bleomycin model has been at the forefront of basic research into the regulation of pulmonary fibrogenesis. The potential of this anti-neoplastic agent to induce experimental lung fibrosis was first appreciated in dogs (28). Later, Snider and colleagues reported and popularized the single-dose intratracheal route of administration in smaller animals (29). Since then, a body of dose–response and time course experiments have determined the amount of drug required to consistently produce a pulmonary fibrotic response when instilled. In addition to the large number of studies detailing growth factor and immunoregulatory changes that occur after bleomycin administration, these studies have also helped characterize changes in extracellular matrix gene expression during fibrotic development (30) as well as the physiologic abnormalities that accompany pulmonary fibrosis (31).
Single doses of bleomycin induce subchronic lesions, but more lasting fibrosis can result from repeated drug dosing (32). Of note, intratracheal instillation produces a bronchiolocentric distribution of fibrosis, while intravenous or intraperitoneal routes of administration characteristically induce subpleural scarring. This versatility in modulating the location of fibrotic lesions offers advantages for studying pulmonary fibrosis with its heterogeneous topography. A similar lesional distribution is encountered in some forms of the idiopathic interstitial pneumonias in man, particularly usual interstitial pneumonia (UIP), the histopathologic equivalent of IPF (33). Other histologic characteristics of bleomycin-induced pulmonary fibrosis also bear resemblance to lesions in human fibrotic lung disease. These include patchy parenchymal inflammation of variable intensity, epithelial cell injury with reactive hyperplasia, basement membrane damage, and interstitial as well as intra-alveolar fibrosis (34). Not infrequently, discrete clusters of spindle-shaped mesenchymal cells may also be apparent within areas of repairing lung (Figure 2)
Figure 2. Histologic features of a murine pulmonary fibroblast–rich lesion with increased extracellular matrix deposition evident 30 d after bleomycin instillation (Masson's trichrome staining).
[More] [Minimize]In the last five years, the bleomycin model has been used to elucidate in vivo mechanisms of TGF-β activation (9), demonstrate the potential of transgenic approaches in modulating the fibrotic response (20), and identify genetic loci governing murine fibrotic susceptibility (36). In terms of assessing antifibrotic therapy, the most obvious success for this model can be attributed to the evaluation of cytokine inhibitors. Crucially, approaches that inhibit the activity of TGF-β using endogenous antagonists (20), antibodies (37), or soluble TGF-β receptors (38) have all demonstrated antifibrotic efficacy in bleomycin-induced pulmonary fibrosis. Moreover, recent studies with fibroblast precursors have shown that this model can elicit progenitor cell recruitment to the lung, indicating its potential for helping to elucidate the mechanisms that control mesenchymal cell trafficking in pulmonary fibrosis (39).
However, caution has also been raised in recent years regarding the validity of bleomycin-induced pulmonary fibrosis as a model of IPF (40). The principal contention relates to two issues: speed of onset (lung remodeling is accelerated in bleomycin-induced pulmonary fibrosis) and durability (bleomycin-induced pulmonary fibrosis in its chronic stages may develop focal emphysema-like changes). In truth, the bleomycin model has never been promoted as an experimental equivalent of IPF. While IPF is a distinct clinico-pathological entity of unknown cause that develops over an indeterminate period of time, bleomycin-induced pulmonary fibrosis reproduces the general morphology of interstitial lung fibrosis without copying any particular lung disorder. The strengths of the bleomycin model lie in its robust reproducibility and versatility as a scaled-down model of general pulmonary fibrosis, itself a collection of disorders unified by a final common pathological pathway of which IPF is only one example.
Major advances in molecular biology and functional genomics have ushered in an unprecedented era in pulmonary fibrosis research. Animal models remain as indispensable now as ever before in driving high-quality hypothesis-based studies. However, the ideal experimental model of pulmonary fibrosis remains elusive. The optimal model should be consistent and incorporate truly progressive fibroproliferative lung repair in a way that is proportionate to the disease intensity and tempo in humans. At the same time, it should be recognized that although many aspects of human pulmonary fibrosis may be mimicked experimentally to some degree of certainty, it may never be possible to develop an animal equivalent of human pulmonary fibrosis.
The instillation of bleomycin has been a leading model of pulmonary fibrosis for many years. Data from bleomycin studies have elucidated many of the biological processes involved in the pathogenesis of pulmonary fibrosis. In recent years, novel information on how lung fibroblasts function as both regulatory and matrix-synthesizing cells in the fibrotic lung has also come from bleomycin studies. However, it is imperative that efforts to develop even better models of the disease continue to be prioritized. The criteria for selecting new models must ideally include the ability to simulate the key events in human lung fibrosis, including inflammation, aberrant epithelial repair, dysregulated fibroblast activity, and transdifferentiation leading to progressive tissue scarring. In the end, the resources invested in developing more clinically relevant models of pulmonary fibrosis can have no better reward than the acquisition of new knowledge allowing therapeutic inroads to be made into a disease with such a dismal prognostic outcome.
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