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Undifferentiated bronchial fibroblasts derived from asthmatic patients display higher elastic modulus than their non-asthmatic counterparts.

Sarna M, Wojcik KA, Hermanowicz P, Wnuk D, Burda K, Sanak M, Czyż J, Michalik M - PLoS ONE (2015)

Bottom Line: Our results demonstrate that asthmatic HBFs form thick and aligned 'ventral' stress fibers accompanied by enlarged focal adhesions.The differences in cytoskeleton architecture between asthmatic and non-asthmatic cells correlate with higher elastic modulus of asthmatic HBFs and their increased predilection to TGF-β-induced FMT.Due to the obvious links between cytoskeleton architecture and mechanical equilibrium, our observations indicate that HBFs derived from asthmatic bronchi can develop considerably higher static tension than non-asthmatic HBFs.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Physics and Biophysics, Faculty of Physics and Applied Computer Science, AGH University of Science and Technology, Krakow, Poland.

ABSTRACT
During asthma development, differentiation of epithelial cells and fibroblasts towards the contractile phenotype is associated with bronchial wall remodeling and airway constriction. Pathological fibroblast-to-myofibroblast transition (FMT) can be triggered by local inflammation of bronchial walls. Recently, we have demonstrated that human bronchial fibroblasts (HBFs) derived from asthmatic patients display some inherent features which facilitate their FMT in vitro. In spite of intensive research efforts, these properties remain unknown. Importantly, the role of undifferentiated HBFs in the asthmatic process was systematically omitted. Specifically, biomechanical properties of undifferentiated HBFs have not been considered in either FMT or airway remodeling in vivo. Here, we combine atomic force spectroscopy with fluorescence microscopy to compare mechanical properties and actin cytoskeleton architecture of HBFs derived from asthmatic patients and non-asthmatic donors. Our results demonstrate that asthmatic HBFs form thick and aligned 'ventral' stress fibers accompanied by enlarged focal adhesions. The differences in cytoskeleton architecture between asthmatic and non-asthmatic cells correlate with higher elastic modulus of asthmatic HBFs and their increased predilection to TGF-β-induced FMT. Due to the obvious links between cytoskeleton architecture and mechanical equilibrium, our observations indicate that HBFs derived from asthmatic bronchi can develop considerably higher static tension than non-asthmatic HBFs. This previously unexplored property of asthmatic HBFs may be potentially important for their myofibroblastic differentiation and bronchial wall remodeling during asthma development.

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Gene expression profile and motility of AS and NA HBFs.A lack of α-SMA (myofibroblast marker) and desmin (muscle cell marker) expression in HBF samples from AS and NA groups (A) was accompanied by a lack of significant differences in the total expression of actin and vinculin between AS and NA samples, as shown by densitometric analysis (B). Human cardiac mesenchymal stromal cells (hcMSC) and TGF-β1-stimulated AS HBFs were used as positive controls for desmin and α-SMA, respectively. Relative optical density (ROD) values represent vinculin and actin levels (mean ± s.d.) calculated for each group by compilation of 4 AS and 4 NA samples, respectively, normalized against GAPDH levels. Time-lapse analyses of cell locomotion did not reveal any significant differences in the motile activity (the total length of displacement and the total length of trajectory) between AS and NA cells (C). Values are means ± s.d. for each group compiled from 4 AS and 4 NA samples, respectively. (*) P < 0.05.
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pone.0116840.g003: Gene expression profile and motility of AS and NA HBFs.A lack of α-SMA (myofibroblast marker) and desmin (muscle cell marker) expression in HBF samples from AS and NA groups (A) was accompanied by a lack of significant differences in the total expression of actin and vinculin between AS and NA samples, as shown by densitometric analysis (B). Human cardiac mesenchymal stromal cells (hcMSC) and TGF-β1-stimulated AS HBFs were used as positive controls for desmin and α-SMA, respectively. Relative optical density (ROD) values represent vinculin and actin levels (mean ± s.d.) calculated for each group by compilation of 4 AS and 4 NA samples, respectively, normalized against GAPDH levels. Time-lapse analyses of cell locomotion did not reveal any significant differences in the motile activity (the total length of displacement and the total length of trajectory) between AS and NA cells (C). Values are means ± s.d. for each group compiled from 4 AS and 4 NA samples, respectively. (*) P < 0.05.

Mentions: It should be emphasized that HBFs were cultivated for relatively short periods of time (16–22 hours) before immunofluorescence and AFM analyses. This limited the influence of ECM deposition on mechanochemical properties of the cells. Moreover, immunoblot analyses of AS and NA HBF populations did not reveal any significant differences in total actin and vinculin levels between the analyzed HBF groups (Fig. 3A, B). A lack of detectable α-SMA and desmin expression in analyzed cells demonstrated that HBFs were undifferentiated and also excluded the presence of smooth muscle cells in the examined samples (Fig. 3A). Moreover, the analyses of cell movement showed no significant differences in the motile activity between AS and NA HBFs (Fig. 3C). This observation is of importance because cell motility can affect the stability of cell adhesion and the size and intracellular distribution of FAs.


Undifferentiated bronchial fibroblasts derived from asthmatic patients display higher elastic modulus than their non-asthmatic counterparts.

Sarna M, Wojcik KA, Hermanowicz P, Wnuk D, Burda K, Sanak M, Czyż J, Michalik M - PLoS ONE (2015)

Gene expression profile and motility of AS and NA HBFs.A lack of α-SMA (myofibroblast marker) and desmin (muscle cell marker) expression in HBF samples from AS and NA groups (A) was accompanied by a lack of significant differences in the total expression of actin and vinculin between AS and NA samples, as shown by densitometric analysis (B). Human cardiac mesenchymal stromal cells (hcMSC) and TGF-β1-stimulated AS HBFs were used as positive controls for desmin and α-SMA, respectively. Relative optical density (ROD) values represent vinculin and actin levels (mean ± s.d.) calculated for each group by compilation of 4 AS and 4 NA samples, respectively, normalized against GAPDH levels. Time-lapse analyses of cell locomotion did not reveal any significant differences in the motile activity (the total length of displacement and the total length of trajectory) between AS and NA cells (C). Values are means ± s.d. for each group compiled from 4 AS and 4 NA samples, respectively. (*) P < 0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4334506&req=5

pone.0116840.g003: Gene expression profile and motility of AS and NA HBFs.A lack of α-SMA (myofibroblast marker) and desmin (muscle cell marker) expression in HBF samples from AS and NA groups (A) was accompanied by a lack of significant differences in the total expression of actin and vinculin between AS and NA samples, as shown by densitometric analysis (B). Human cardiac mesenchymal stromal cells (hcMSC) and TGF-β1-stimulated AS HBFs were used as positive controls for desmin and α-SMA, respectively. Relative optical density (ROD) values represent vinculin and actin levels (mean ± s.d.) calculated for each group by compilation of 4 AS and 4 NA samples, respectively, normalized against GAPDH levels. Time-lapse analyses of cell locomotion did not reveal any significant differences in the motile activity (the total length of displacement and the total length of trajectory) between AS and NA cells (C). Values are means ± s.d. for each group compiled from 4 AS and 4 NA samples, respectively. (*) P < 0.05.
Mentions: It should be emphasized that HBFs were cultivated for relatively short periods of time (16–22 hours) before immunofluorescence and AFM analyses. This limited the influence of ECM deposition on mechanochemical properties of the cells. Moreover, immunoblot analyses of AS and NA HBF populations did not reveal any significant differences in total actin and vinculin levels between the analyzed HBF groups (Fig. 3A, B). A lack of detectable α-SMA and desmin expression in analyzed cells demonstrated that HBFs were undifferentiated and also excluded the presence of smooth muscle cells in the examined samples (Fig. 3A). Moreover, the analyses of cell movement showed no significant differences in the motile activity between AS and NA HBFs (Fig. 3C). This observation is of importance because cell motility can affect the stability of cell adhesion and the size and intracellular distribution of FAs.

Bottom Line: Our results demonstrate that asthmatic HBFs form thick and aligned 'ventral' stress fibers accompanied by enlarged focal adhesions.The differences in cytoskeleton architecture between asthmatic and non-asthmatic cells correlate with higher elastic modulus of asthmatic HBFs and their increased predilection to TGF-β-induced FMT.Due to the obvious links between cytoskeleton architecture and mechanical equilibrium, our observations indicate that HBFs derived from asthmatic bronchi can develop considerably higher static tension than non-asthmatic HBFs.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Physics and Biophysics, Faculty of Physics and Applied Computer Science, AGH University of Science and Technology, Krakow, Poland.

ABSTRACT
During asthma development, differentiation of epithelial cells and fibroblasts towards the contractile phenotype is associated with bronchial wall remodeling and airway constriction. Pathological fibroblast-to-myofibroblast transition (FMT) can be triggered by local inflammation of bronchial walls. Recently, we have demonstrated that human bronchial fibroblasts (HBFs) derived from asthmatic patients display some inherent features which facilitate their FMT in vitro. In spite of intensive research efforts, these properties remain unknown. Importantly, the role of undifferentiated HBFs in the asthmatic process was systematically omitted. Specifically, biomechanical properties of undifferentiated HBFs have not been considered in either FMT or airway remodeling in vivo. Here, we combine atomic force spectroscopy with fluorescence microscopy to compare mechanical properties and actin cytoskeleton architecture of HBFs derived from asthmatic patients and non-asthmatic donors. Our results demonstrate that asthmatic HBFs form thick and aligned 'ventral' stress fibers accompanied by enlarged focal adhesions. The differences in cytoskeleton architecture between asthmatic and non-asthmatic cells correlate with higher elastic modulus of asthmatic HBFs and their increased predilection to TGF-β-induced FMT. Due to the obvious links between cytoskeleton architecture and mechanical equilibrium, our observations indicate that HBFs derived from asthmatic bronchi can develop considerably higher static tension than non-asthmatic HBFs. This previously unexplored property of asthmatic HBFs may be potentially important for their myofibroblastic differentiation and bronchial wall remodeling during asthma development.

Show MeSH
Related in: MedlinePlus