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Modeling fibrosis using fibroblasts isolated from scarred rat vocal folds

View Article: PubMed Central - PubMed

ABSTRACT

Following injury, pathologically activated vocal fold fibroblasts (VFFs) can engage in disordered extracellular matrix (ECM) remodeling, leading to VF fibrosis and impaired voice function. Given the importance of scar VFFs to phenotypically appropriate in vitro modeling of VF fibrosis, we pursued detailed characterization of scar VFFs obtained from surgically injured rat VF mucosae, compared to those obtained from experimentally naïve, age-matched tissue. Scar VFFs initially exhibited a myofibroblast phenotype characterized by increased proliferation, increased Col1a1 transcription and collagen, type I synthesis, increased Acta2 transcription and α-smooth muscle actin synthesis, and enhanced contractile function. These features were most distinct at passage 1 (P1); we observed a coalescence of the scar and naïve VFF phenotypes at later passages. An empirical Bayes statistical analysis of the P1 cell transcriptome identified 421 genes that were differentially expressed by scar, compared to naïve, VFFs. These genes were primarily associated with the wound response, ECM regulation, and cell proliferation. Follow-up comparison of P1 scar VFFs and their in vivo tissue source showed substantial transcriptomic differences. Finally, P1 scar VFFs responded to treatment with hepatocyte growth factor and transforming growth factor-β3, two biologics with reported therapeutic value. Despite the practical limitations inherent to working with early passage cells, this experimental model is easily implemented in any suitably equipped laboratory and has the potential to improve the applicability of preclinical VF fibrosis research.

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Effect of growth factor stimulation on scar-related transcriptional activityExogenous HGF stimulation downregulated Col1a1 (a) and Acta2 (b) transcription in scar VFFs at P1 in a dose-dependent manner, whereas exogenous TGF-β3 stimulation upregulated Acta2 transcription only (b). Data are presented as mean fold change ± s.e.m. versus the untreated control (Ctl) condition and are log2-transformed to best represent bidirectional stimulation effects. All experiments were performed with n = 4 biological replicates per condition. *, p < 0.05.
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Figure 6: Effect of growth factor stimulation on scar-related transcriptional activityExogenous HGF stimulation downregulated Col1a1 (a) and Acta2 (b) transcription in scar VFFs at P1 in a dose-dependent manner, whereas exogenous TGF-β3 stimulation upregulated Acta2 transcription only (b). Data are presented as mean fold change ± s.e.m. versus the untreated control (Ctl) condition and are log2-transformed to best represent bidirectional stimulation effects. All experiments were performed with n = 4 biological replicates per condition. *, p < 0.05.

Mentions: Given our data showing clear phenotypic differences between naïve and scar VFFs at P1, we evaluated the responsiveness of scar VFFs to stimulation with exogenous HGF and TGF-β3. These biologics have shown therapeutic potential when delivered to naïve VFFs in vitro,17,29,47,48 as well as when delivered to injured or scarred VF mucosae in vivo.17,30,49–51 Treatment with HGF downregulated Col1a1 and Acta2 transcription in a dose-dependent manner (p < .05; Figure 6a,b). Treatment with TGF-β3 had no effect on Col1a1 (p > .05; Figure 6a) but upregulated Acta2 transcription at all doses (p < .05; Figure 6b). These data show that P1 scar VFFs are amenable to manipulation using growth factors, as has been reported for their naïve counterparts.


Modeling fibrosis using fibroblasts isolated from scarred rat vocal folds
Effect of growth factor stimulation on scar-related transcriptional activityExogenous HGF stimulation downregulated Col1a1 (a) and Acta2 (b) transcription in scar VFFs at P1 in a dose-dependent manner, whereas exogenous TGF-β3 stimulation upregulated Acta2 transcription only (b). Data are presented as mean fold change ± s.e.m. versus the untreated control (Ctl) condition and are log2-transformed to best represent bidirectional stimulation effects. All experiments were performed with n = 4 biological replicates per condition. *, p < 0.05.
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Related In: Results  -  Collection

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Figure 6: Effect of growth factor stimulation on scar-related transcriptional activityExogenous HGF stimulation downregulated Col1a1 (a) and Acta2 (b) transcription in scar VFFs at P1 in a dose-dependent manner, whereas exogenous TGF-β3 stimulation upregulated Acta2 transcription only (b). Data are presented as mean fold change ± s.e.m. versus the untreated control (Ctl) condition and are log2-transformed to best represent bidirectional stimulation effects. All experiments were performed with n = 4 biological replicates per condition. *, p < 0.05.
Mentions: Given our data showing clear phenotypic differences between naïve and scar VFFs at P1, we evaluated the responsiveness of scar VFFs to stimulation with exogenous HGF and TGF-β3. These biologics have shown therapeutic potential when delivered to naïve VFFs in vitro,17,29,47,48 as well as when delivered to injured or scarred VF mucosae in vivo.17,30,49–51 Treatment with HGF downregulated Col1a1 and Acta2 transcription in a dose-dependent manner (p < .05; Figure 6a,b). Treatment with TGF-β3 had no effect on Col1a1 (p > .05; Figure 6a) but upregulated Acta2 transcription at all doses (p < .05; Figure 6b). These data show that P1 scar VFFs are amenable to manipulation using growth factors, as has been reported for their naïve counterparts.

View Article: PubMed Central - PubMed

ABSTRACT

Following injury, pathologically activated vocal fold fibroblasts (VFFs) can engage in disordered extracellular matrix (ECM) remodeling, leading to VF fibrosis and impaired voice function. Given the importance of scar VFFs to phenotypically appropriate in vitro modeling of VF fibrosis, we pursued detailed characterization of scar VFFs obtained from surgically injured rat VF mucosae, compared to those obtained from experimentally na&iuml;ve, age-matched tissue. Scar VFFs initially exhibited a myofibroblast phenotype characterized by increased proliferation, increased Col1a1 transcription and collagen, type I synthesis, increased Acta2 transcription and &alpha;-smooth muscle actin synthesis, and enhanced contractile function. These features were most distinct at passage 1 (P1); we observed a coalescence of the scar and na&iuml;ve VFF phenotypes at later passages. An empirical Bayes statistical analysis of the P1 cell transcriptome identified 421 genes that were differentially expressed by scar, compared to na&iuml;ve, VFFs. These genes were primarily associated with the wound response, ECM regulation, and cell proliferation. Follow-up comparison of P1 scar VFFs and their in vivo tissue source showed substantial transcriptomic differences. Finally, P1 scar VFFs responded to treatment with hepatocyte growth factor and transforming growth factor-&beta;3, two biologics with reported therapeutic value. Despite the practical limitations inherent to working with early passage cells, this experimental model is easily implemented in any suitably equipped laboratory and has the potential to improve the applicability of preclinical VF fibrosis research.

No MeSH data available.


Related in: MedlinePlus