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Myofibroblast or Smooth Muscle

Do In Vitro Systems Adequately Replicate Tissue Smooth Muscle?

National Jewish Medical and Research Center, Denver, Colorado and University of Colorado Health Sciences Center, Denver, Colorado

In this issue of the Journal (pp. 367–372 and pp. 379–385), two different investigative groups put forth intriguing hypotheses to support a primary role for the airway smooth muscle (ASM) cell in asthma pathogenesis (1, 2). The original investigation by Chan and colleagues (1) suggests that asthmatic ASM cells produce more eotaxin in response to interaction with asthmatic extracellular matrix than do normal ASM cells, thereby contributing to enhanced eosinophilic inflammation. The second, a Pulmonary Perspective review article by Borger and colleagues (2), describes mounting evidence from many studies to support one or more abnormalities in ASM cells, which could make them both an initiator and sustainer of the asthma phenotype. While these are both provocative pieces, and the use of primary human cells should always be considered laudatory, a word of caution related to all in vitro studies of "airway smooth muscle cells" is needed.

As noted in the Perspective by Borger and colleagues, numerous recent in vitro studies of ASM cells have reported obtaining these from human lung tissue, including autopsies, surgical resections, or, in most studies of asthmatic ASM, endobronchial biopsies. The studies all describe obtaining such cells by isolating smooth muscle bundles free from surrounding tissue using a dissecting microscope (not using the more precise but cell death–inducing laser capture technique) and then placing them into cell culture. After growth in fetal bovine serum and subsequent passaging (often up to 10 times), the cells are identified as smooth muscle cells on the basis of their expression of -smooth muscle actin, calponin, or desmin (3). The cells are then stimulated in a variety of ways, numerous readouts are obtained, and the results are reported as specific for ASM cells.

Several caveats should be addressed regarding the specificity of these "smooth muscle cell" culture systems before results using such techniques are considered to be definitive findings:

1. Dissecting pure smooth muscle from large, unfixed pieces of tissue is difficult. Precise dissection of smooth muscle bundles from endobronchial biopsies (often < 1 mm³ in volume and usually the predominant source for asthmatic ASM cells) is likely to be nearly impossible. ASM cells are not obviously different from surrounding matrix in the endobronchial biopsy pieces and no stains/markers are used to specifically identify these cells. Dissecting out smooth muscle bundles, even in the best of cases, is not likely to yield pure smooth muscle cells. Fibroblasts are in close proximity (and may even be intermingled with) ASM cells such that any dissection probably will yield a mix of cell types, including fibroblasts.
   
2. Fibroblasts, grown in the presence of serum and with successive passages, can become myofibroblasts and produce large quantities of -smooth muscle actin, one of the most commonly used "ASM cell" markers. In addition, myofibroblasts can produce calponin, desmin, and numerous other putative ASM cell markers, such that it is virtually impossible to distinguish them from true ASM cells.
   
3. ASM cells, even when relatively pure, lose their contractile phenotype in the presence of serum (3). ASM cells are described as differentiating into a "synthetic" phenotype that is virtually interchangeable with a myofibroblast. They do not form smooth muscle bundles in vitro, one of the prime differentiators of ASM cells and myofibroblasts in vivo (4).
   
4. The reported studies often compare asthmatic "ASM cells" to normal ASM cells. However, the normal ASM cells are generally obtained from surgical resections or autopsies, whereas the asthmatic ASM cells are obtained primarily from endobronchial biopsies. As noted in the first concern, although both tissue samples are likely to be a mix of different cell types, it is more likely that endobronchial specimens will be highly "contaminated" with fibroblasts than will the cells from the surgical tissue. Second, it is also probable that the airway region in which the smooth muscle cells are obtained in the surgical specimens is more distal than that of the proximal airway biopsies. Even if these are purely comparisons of ASM cells, these cells are likely to be obtained from different regions. A recent study from our laboratory suggests that fibroblasts from the distal lung are phenotypically quite different from proximal airway fibroblasts, including the observation that distal lung fibroblasts produce significantly less eotaxin than proximal airway fibroblasts (5). Whether similar differences could exist for the "ASM cell" studies (and even partly explain the results of Chan and colleagues) is not clear.
   

Keeping these issues in mind, is it, in fact, important to differentiate between smooth muscle cells, fibroblasts, or myofibroblasts in in vitro culture systems? For some studies, such as the one in this issue by Chan and colleagues, the implications are not fully dependent on determining precisely which cell type contributes to the differences reported. Fibroblasts and myofibroblasts can just as easily produce eotaxin to attract eosinophils and contribute to inflammation in the airway wall (6). We are not aware of any studies that suggest eosinophils are increased specifically in smooth muscle, so specifically identifying ASM cells as the source of the eotaxin is not critical. Some of the studies referred to by Borger and colleagues, and particularly those which suggest that properties observed in the cultured ASM cells contribute to the specific abnormalities observed in vivo, are more concerning (7–9). In cases such as these, determining that the findings observed in the "ASM cells" specifically differ from those which utilize cultured fibroblasts and/or myofibroblasts obtained from the surrounding tissue are urgently needed. For studies with such smooth muscle–specific implications, parallel cultures should be set up using these other cell types to determine whether the results are, in fact, specific for the cells believed to represent ASM. Until such studies are forthcoming, it is impossible to determine the importance of either the properties of the cultured "ASM cells" or their implications for the differences observed in smooth muscle in the lungs of patients with asthma. Such comparisons are strongly encouraged.

FOOTNOTES

Conflict of Interest Statement: Neither of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

REFERENCES

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4. Seaton A, Leitch AG, Seaton A. In: Seaton A, editor. Crofton and Douglas' respiratory diseases, version 3, Vol. 1. Oxford, UK: Blackwell; 2000. p. 13.
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