Characterization of human vocal fold fibroblasts

Introduction Vocal fold fibroblasts (VFF) are the central cell type of the vocal fold (VF) lamina propria and play a central role under physiological conditions but also after VF injury and during fibrogenesis. Little is known about the behavior of human VFF (hVFF) from injured or normal tissue, as these have been inaccessible so far. Knowledge of pathways of scar formation and changes in the extracellular matrix (ECM) following VF injury derived mostly from studies in canine, ferret, porcine and rabbit models. The next step is to validate these findings with hVFF. Aims The overall objective of the project is to better characterize hVFF from scarred and normal VF in a larger sample size (n=8). We will test the hypothesis that hVFF derived from scarred and normal VF differ morphologically, in growth characteristics, production of extracellular matrix (ECM) components and in gene expression. Procedures In cooperation with Prof. Stellan Hertegård from Karolinska Institute, Department of Clinical Science, Intervention and Technology, Division of Ear, Nose and Throat Diseases, we plan to nearer specify characteristics of hVFF. During previous interventions at the host institution bilateral VF specimens (n=8) were obtained and stored deep frozen. Informed consent by all patients and approval by the local Ethics committee was obtained. Fibroblasts will be extracted from all deep-frozen specimens according to established protocols. In different work packages we will explore morphological and growth characteristics, expression patterns of several extracellular matrix (ECM) components and gene expression. Analyses comprise quantitative (Western blot and real time polymerase chain reaction) and qualitative (immunocytochemistry) methods, as well as microarray analyses for gene expression. Criteria for success are the different amounts of produced ECM components, cytokines and clusters of up- and down-regulated genes. These will be compared inter- and intra-individually. Data will be interpreted under special consideration of data obtained from medical records. Innovations of the proposed project This innovative project represents the first attempt to characterize hVFF from scarred and un-injured VF in a large series of cases. Our work will contribute significantly to the basic knowledge in laryngeal cell biology. It will create a solid basis for further exploration of the cellular and molecular mechanisms of hVFF and their underlying function in health and injury. Human VFF are a central target for future development of treatment strategies and need to be nearer specified. This needs to be done in larger series, as primary cell lines exhibit considerable variations.

Vocal fold fibroblasts (VFF) represent the main cell type of the vibrating part of the laryngeal vocal fold (VF) mucosa. Their main function is to provide production of certain proteins that guarantee pliability and thus proper vibration characteristics of the VF. Chronic VF scars are a condition that is accompanied with a lifelong hoarse and breathy voice which has a considerable effect on quality of life in affected patients. Underlying reasons for this are chronic inflammations or trauma to the VF by surgery or prolonged intubation. Due to the limited access to human VFF little is known about the underlying pathologies of chronic VF scars. Aim of the project was to characterize human VFF which were gathered from chronic VF scar and healthy VF tissue from the same donor. Microscopical analysis did not show morphological analysis between normal and scarred VFF, neither did growth patterns. On the genetic level scarred VFF showed increased metabolism under stimulation with an antifibrotic agent (HGF hepatocyte growth factor). This means that even years after initial injury scarred cells are responsive to treatment. An analysis of cell surface receptors (flow cytometry) did not show evidence for presence of the so called c-met receptor that is considered to be the connecting site for HGF. This might indicate that in VFF another mechanism of HGF activation might be present. An ongoing project will explore the secretion of numerous proteins by normal and scar VFF (proteomics).