Modeling fibrosis using fibroblasts isolated from scarred rat vocal folds

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.

Alterations in macrophage polarization in injured murine vocal folds

“…Thus, the inflammatory phase is a key phase in initiating and controlling wound‐healing cascades, and monocyte lineage cells are thought to assume a large role in this phase. However, most previous studies in vocal fold biology have focused on the behaviors or phenotypes of the vocal fold fibroblasts because they primarily synthesize the extracellular matrix (ECM) components affecting the biomechanical properties of the vocal fold mucosa …”

Characterization of aged rat vocal fold fibroblasts

“…Vocal fold fibroblasts (VFFs) are thought to play major roles in age‐related changes, because extracellular matrix (ECM) is primarily produced by the VFFs. Fibroblasts are widely distributed in many organs, but their characteristics and manner of aging could change depending on their origin or biological conditions . Few studies directly compared aged VFFs with young VFFs in their naïve condition, and only the following characteristics were implied in aged VFFs: impaired proliferative capacity, reduced hyaluronan production, and maintained or reduced collagen production.…”

“…The discrepancy between in vitro collagen production and histological deposition was explained by elongated turnover time, which has not been tested before. Moreover, myofibroblasts in aged VFFs have never been estimated, though their increase was histologically confirmed, and they are thought to produce an increased amount of type I collagen …”

“…Fibroblasts are widely distributed in many organs, but their characteristics and manner of aging could change depending on their origin or biological conditions. [11][12][13][14][15] Few studies directly compared aged VFFs with young VFFs in their naïve condition, and only the following characteristics were implied in aged VFFs 8,16 : impaired proliferative capacity, reduced hyaluronan production, and maintained or reduced collagen production. The discrepancy between in vitro collagen production and histological deposition was explained by elongated turnover time, which has not been tested before.…”

“…Moreover, myofibroblasts in aged VFFs have never been estimated, though their increase was histologically confirmed, and they are thought to produce an increased amount of type I collagen. 7,15 The clarification of the aging physiology of vocal folds is necessary prior to the development of presbyphonia treatments. We hypothesized that age-related cell-cycle arrest would affect cell proliferation, and that myofibroblast population would play some role in aged VFFs.…”