Abstract
ObjectiveTo gain insight into the molecular mechanisms underlying the early stages of vocal fold extracellular matrix (ECM) remodeling after a mid-membranous injury resulting from the use of human amniotic epithelial cells (hAEC), as a novel regenerative medicine cell-based therapy.MethodsVocal folds of six female, New Zealand White rabbits were bilaterally injured. Three rabbits had immediate bilateral direct injection of 1 × 106 hAEC in 100 µl of saline solution (hAEC) and three with 100 µl of saline solution (controls, CTR). Rabbits were euthanized 6 weeks after injury. Proteomic analyses (in-gel trypsin protein digestion, LC–MS/MS, protein identification using Proteome Discoverer and the Uniprot Oryctolagus cuniculus (Rabbit) proteome) and histological analyses were performed.ResultshAEC treatment significantly increased the expression of ECM proteins, elastin microfibril interface-located protein 1 (EMILIN-1) and myocilin that are primarily involved in elastogenesis of blood vessels and granulation tissue. A reactome pathway analysis showed increased activity of the anchoring fibril formation by collagen I and laminin, providing mechanical stability and activation of cell signaling pathways regulating cell function. hAEC increased the abundance of keratin 1 indicating accelerated induction of the differentiation programming of the basal epithelial cells and, thereby, improved barrier function. Lastly, upregulation of Rab GDP dissociation inhibitor indicates that hAEC activate the vesicle endocytic and exocytic pathways, supporting the exosome-mediated activation of cell–matrix and cell-to-cell interactions.ConclusionsThis pilot study suggests that injection of hAEC into an injured rabbit vocal fold favorably alters ECM composition creating a microenvironment that accelerates differentiation of regenerated epithelium and promotes stabilization of new blood vessels indicative of accelerated and improved repair.
Highlights
Vocal fold trauma is a relatively frequent event caused by mechanical injury, prolonged inflammation, radiotherapy, or surgery
The vocal fold consists of three main layers: the layer of epithelial cells with non-keratinized stratified squamous phenotype on the luminal side, which forms the barrier with the external environment; the underlying lamina propria, which provides strength and elasticity/ compliance, and the deep vocalis muscle
Lamina propria is restored by development of granulation tissue involving differentiation of fibroblasts into extracellular matrix (ECM) producing contractile myofibroblasts coupled with revascularization [4]
Summary
Vocal fold trauma is a relatively frequent event caused by mechanical injury, prolonged inflammation, radiotherapy, or surgery. Upon injury of the vocal fold, the epithelial layer is restored by migration and proliferation of epithelial cells that are adjacent to the wound site (reepithelization). This leads to wound closure which is followed by epithelial differentiation and stratification to establish barrier function [3]. There is a continuous bidirectional collaboration between fibroblasts, epithelial, endothelial, immune, and other cell types, as well as between cells and their surrounding ECM molecules, termed dynamic reciprocity This involves regulation of cell function by biochemical and mechanical signals from ECM molecules through specific receptor-ligand interactions, which in turn influences the composition and quantity of the ECM produced by the cells [4]. Resolution of the initial inflammatory process and wound closure is critical to prevent excessive disordered deposition and subsequent inadequate remodeling of ECM in the lamina propria (fibrosis), thereby preventing stiffness and impaired vibratory properties
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