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The Myofibroblast in Pulmonary Fibrosis^*

^* From the Department of Pathology, University of Michigan, Ann Arbor, MI.

Correspondence to: Sem H. Phan, PhD, MD, Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109-0602; e-mail: shphan{at}umich.edu

	Abstract

TOP Abstract Introduction Pulmonary Fibrosis and the... Myofibroblast Differentiation Myofibroblast Disappearance Conclusion References

 
The pathogenesis of pulmonary fibrosis remains incompletely understood. Studies of associated inflammation have led to the discovery of a number of cytokines and chemokines that are found to be important either directly or indirectly for the fibrotic process. However, the importance of inflammation in pulmonary fibrosis is unclear, and at the time of diagnosis the inflammatory component is variable and usually not responsive to anti-inflammatory therapeutic agents. Patients usually exhibit evidence of active fibrosis with increased numbers of activated fibroblasts, many of which have the phenotypic characteristics of myofibroblasts. At these sites, increased amounts of extracellular matrix deposition are evident with effacement of the normal alveolar architecture. Animal model studies show the myofibroblast to be the primary source of type I collagen gene expression in active fibrotic sites. In vitro studies show differentiation of these cells from fibroblasts under the influence of certain cytokines but indicate their susceptibility to nitric oxide-mediated apoptosis. In addition to promoting myofibroblast differentiation, transforming growth factor-ß1 provides protection against apoptosis. Thus, this well-known fibrogenic cytokine is important both for the emergence of the myofibroblast and its survival against apoptotic stimuli. This is consistent with the critical importance of this cytokine in diverse models of fibrosis in various tissues. In view of these properties, the persistence or prolonged survival of the myofibroblast may be key to understanding why certain forms of lung injury may result in progressive disease, terminating in end-stage disease.

Key Words: apoptosis • collagen • fibroblasts • myofibroblasts • telomerase • transforming growth factor-ß1

	Introduction

TOP Abstract Introduction Pulmonary Fibrosis and the... Myofibroblast Differentiation Myofibroblast Disappearance Conclusion References

 
Although pulmonary fibrosis has diverse etiologies, there is a common feature characteristic of this process, namely, the abnormal deposition of extracellular matrix that effaces the normal lung tissue architecture. A key cellular source of this matrix is the mesenchymal cell population that occupies much of the fibrotic lesion during the active period of fibrosis. This population is heterogeneous with respect to a number of key phenotypes. One of these phenotypes is the myofibroblast, which is commonly identified by its expression in -smooth muscle actin and by features that are intermediate between the bona fide smooth muscle cell and the fibroblast. The de novo appearance of myofibroblasts at sites of wound healing and tissue repair/fibrosis is associated with the period of active fibrosis and is considered to be involved in wound contraction. Furthermore, the localization of myofibroblasts at sites undergoing active extracellular matrix deposition suggests an important role for these cells in the genesis of the fibrotic lesion. In recognition of the potential importance of this cell in fibrosis, and perhaps in its persistence or progression, studies have focused on the nature and precise role or roles of this cell in the context of pulmonary fibrosis.

	Pulmonary Fibrosis and the Myofibroblast

TOP Abstract Introduction Pulmonary Fibrosis and the... Myofibroblast Differentiation Myofibroblast Disappearance Conclusion References

 
The presence of myofibroblasts in patients with pulmonary fibrosis is amply documented in both lung tissues taken from patients with pulmonary fibrosis as well as in those taken from animal models of the disease.^{1 2 3 4} Since fibroblasts in fibrotic lesions are considered historically to be the cells responsible for the deposition of the matrix that constitutes the scar, early studies focused on the importance of the myofibroblast in this capacity. Thus, localization at sites undergoing active matrix deposition and the elevated collagen synthetic capacity of granulation tissue myofibroblasts strongly suggest such a role for the myofibroblast in fibrosis.³ Direct confirmation that these cells are key sources of collagen gene expression is provided by the use of combined in situ hybridization for procollagen type I messenger RNA and immunostaining for -smooth muscle actin.⁵ Using similar approaches, however, these cells were additionally found to be significant sources of several cytokines, including transforming growth factor (TGF)-ß1, a well-established key fibrogenic mediator, and the CC chemokine, monocyte chemotactic protein-1.^{6 7} Given the inflammatory properties of both of these cytokines, the myofibroblast appears to play additional potentially important roles beyond the deposition of extracellular matrix. By elaborating on these mediators, they have the potential of contributing to the recruitment of inflammatory cells, and thus of intensifying or prolonging the inflammation that is often associated with fibrosis. Such amplification of the inflammatory response may result in a positive feedback loop resulting in the intensification and progression of fibrosis.

An additional property of the myofibroblast is its contractility. This is thought to be important in wound contraction and perhaps to contribute to the altered mechanical characteristics of the fibrotic lung.^{1 2} This property can be observed in vitro as the contraction of myofibroblast-populated collagen gels, which positively correlates with increased -smooth muscle actin expression, a marker of myofibroblast differentiation.⁸ Thus, this cell appears to have the capability of reproducing important features of fibrotic lung tissue.

From these studies, it appears that the myofibroblast has the potential to play important roles in the pathogenesis of pulmonary fibrosis. Its dual role, as a key source of extracellular matrix and as an inflammatory cell, makes it the key cell in two processes that represent the hallmark of pulmonary fibrosis. Its importance in altering the compliance of the lung in this disease provides additional support for its role in pathogenesis. Further support for these roles is provided by the correlation between the appearance of the myofibroblast and active fibrosis, as manifested by increased cellularity, and heightened collagen and cytokine gene expression. The distinct kinetics of the appearance and disappearance of the myofibroblast in normal wound healing and self-limiting models of pulmonary fibrosis parallels the initiation of active fibrosis and its resolution or termination.^{5 9} The failure of the disease to resolve, as seen in patients with progressive disease, correlates with the persistence of the myofibroblast.³ These characteristics are reminiscent of the behavior of inflammatory cells at sites of tissue injury and inflammation, thus further supporting the designation of the myofibroblast as an inflammatory cell. In view of this similarity to the initiation and termination of inflammation, plus the evidence for the importance of the myofibroblast, recent studies have focused on the mechanisms underlying its de novo appearance and its disappearance.

	Myofibroblast Differentiation

TOP Abstract Introduction Pulmonary Fibrosis and the... Myofibroblast Differentiation Myofibroblast Disappearance Conclusion References

 
Since there are significant parallels between inflammatory cells in inflammation and myofibroblasts in fibrosis, it is not surprising to note that fibroblasts can be chemotactically recruited. Additionally recruited inflammatory cells undergo distinct phenotypic changes, as exemplified by the differentiation of monocytes to macrophages. With respect to the myofibroblast, a key marker of its differentiation is the expression of -smooth muscle actin accompanied by heightened collagen and cytokine gene expression, increased contractility, and reduced motility and proliferative capacity. Morphologically, this cell contains dense aggregates of microfilaments and exhibits specialized contacts with adjacent myofibroblasts and extracellular matrix components.^{10 11 12} The cytoskeletal composition is somewhat variable, depending on the tissue localization. Thus, myofibroblasts in the fibrotic lung express -smooth actin and vimentin, but do not express desmin, except for those myofibroblasts that are localized in more peripheral and subpleural areas of fibrosis.⁵ In a self-limiting model of lung injury and fibrosis, they emerge during the proliferative and active phase of fibrosis, and subsequently gradually disappear.⁵ The origin of these myofibroblasts is controversial, but kinetic studies suggest that the myofibroblast in pulmonary fibrosis is derived from preexisting peribronchial and perivascular adventitial fibroblasts.⁵ This observation is supported by in vitro biochemical and morphologic evidence of the fibroblastic origin of the myofibroblast.^{13 14 15} Furthermore, cultured fibroblasts in vitro can be induced to differentiate into myofibroblasts by treatment with cytokines, such as TGF-ß and interleukin (IL)-4.^{16 17} Thus, with respect to pulmonary fibrosis, the presumed mechanism for the emergence of the myofibroblast is its cytokine-induced differentiation from fibroblasts, the population of which has been expanded under the influence of growth factors secreted by inflammatory and other lung cells.

This paradigm is complicated by the discovery of an additional activated lung fibroblast phenotype in a model of lung injury and fibrosis. This phenotype is characterized by the expression of telomerase activity, which does not localize to cells expressing -smooth muscle actin (ie, in nonmyofibroblasts).¹⁸ This seems to suggest that the telomerase-positive cells represent an intermediate activated phenotype between the quiescent fibroblast and the highly activated myofibroblast. However, whether this telomerase phenotype is an obligatory intermediate step in myofibroblast differentiation remains to be determined.

Myofibroblast differentiation can be defined on the basis of the induction of -smooth muscle actin expression. The significance of the expression of this actin isoform vis-à-vis the other more functionally relevant (to fibrogenesis) phenotypic features of the myofibroblast are unclear. However, given the unique nature of this induction of -smooth muscle actin as a differentiation marker, studying the mechanism regulating the induction of this gene may provide insight into the means of clarifying the differentiation process. Analysis of the effects of the differentiation-promoting cytokine TGF-ß on the -smooth muscle actin promoter shows some unique features that are distinguishable from those seen in smooth muscle cells.¹⁹ In contrast to smooth muscle cells and certain cell lines, the induction of -smooth muscle actin expression in myofibroblast differentiation requires only a TGF-ß control element, while the CArG elements present in the promoter are unnecessary. A requirement for additional elements and regulatory factors requires further investigation. The distinction from smooth muscle and other cells of this unique regulatory mechanism in myofibroblast differentiation suggests a potential significance for future attempts at arresting the progression of fibrosis by the inhibition of this process.

	Myofibroblast Disappearance

TOP Abstract Introduction Pulmonary Fibrosis and the... Myofibroblast Differentiation Myofibroblast Disappearance Conclusion References

 
In normal wound healing, the number of myofibroblasts gradually declines as the healing process is successfully completed.^{9 11 12} Similarly, in a self-limiting model of pulmonary fibrosis, myofibroblasts gradually disappear as the active fibrotic phase is terminated.⁵ In contrast, these cells persist and can be found in various stages of human pulmonary fibrosis where the disease is progressive.³ Thus, the mechanism of the myofibroblast disappearance is of potential interest since it can provide insight into the basis for its persistence and hence into the maintenance or progression of the fibrosis.

Studies of wound healing²⁰ suggest that myofibroblast disappearance occurs via apoptosis. Support for a similar mechanism in studies of self-limiting pulmonary fibrosis in rodents is provided by in vitro evidence showing increased susceptibility of lung myofibroblasts for undergoing apoptosis relative to fibroblasts.²¹ Apoptosis induced by IL-1ß is dependent on the induction of inducible nitric oxide synthase (iNOS) expression and nitric oxide production by fibroblasts exclusively, while the target myofibroblasts themselves do not express iNOS.²² Furthermore, the apoptotic pathway involves a reduction in the antiapoptotic protein Bcl-2 without affecting the proapoptotic protein Bax. In addition to inducing myofibroblast differentiation from fibroblasts, TGF-ß1 promotes myofibroblast survival by affording protection against IL-1ß apoptosis by inhibiting iNOS induction and the apoptosis-associated reduction in Bcl-2 expression.²² Despite this evidence of in vitro susceptibility to IL-1ß-induced apoptosis, the actual in vivo signal for myofibroblast apoptosis is unknown. A possible clue is provided by the parallels between inflammatory cells and myofibroblasts as noted earlier. In the case of inflammatory cells, it appears that the loss of growth factor signaling represents a key trigger for apoptosis. In the case of eosinophils, the loss of IL-5 signaling induces apoptosis.²³ In view of the multiple functions of TGF-ß in promoting myofibroblast differentiation and maintaining its survival, a loss of TGF-ß signaling may represent a signal for myofibroblast apoptosis. This possibility has some support in vivo, wherein the gradual decline in myofibroblast numbers occurs at a period when TGF-ß expression is also declining in cases of bleomycin-induced pulmonary fibrosis.^{5 6}

	Conclusion

TOP Abstract Introduction Pulmonary Fibrosis and the... Myofibroblast Differentiation Myofibroblast Disappearance Conclusion References

 
The emergence and disappearance of the myofibroblast appears to correlate with the initiation of active fibrosis and its resolution, respectively. In addition, the myofibroblast has many phenotypic features, which embody much of the pathologic alterations in fibrotic lung tissue. These features would seem to argue for an important role for the myofibroblast in the pathogenesis of pulmonary fibrosis. Furthermore, the persistence of the myofibroblast may herald progressive disease, and, conversely, its disappearance may be an indicator of resolution. This in turn suggests that future therapeutic strategies targeting the myofibroblast may be productive. Clearly, more studies are needed to uncover the regulatory mechanisms involved in myofibroblast differentiation and apoptosis before such strategies can be evaluated.

	Footnotes

 
Abbreviations: IL = interleukin; iNOS = inducible nitric oxide synthase; TGF = transforming growth factor

	References

TOP Abstract Introduction Pulmonary Fibrosis and the... Myofibroblast Differentiation Myofibroblast Disappearance Conclusion References

 

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