Date of Award

5-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Bioengineering

Committee Member

Dr. Ken Webb, Committee Chair

Committee Member

Dr. Jeoung Soo Lee

Committee Member

Dr. Jeremy L. Barth

Committee Member

Dr. Jiro Nagatomi

Abstract

Extracellular matrix (ECM) is a dynamic and complex environment characterized by biophysical, mechanical and biochemical properties specific for each tissue. Cells constantly experience dynamic mechanical loadings that include compression, shear, tension, hydrostatic pressure, and interstitial fluid flow. Through the process of mechano-chemical conversion, mechanical stimulation activates intracellular biochemical signaling that affects many aspects of cell behavior including cell proliferation and differentiation, as well as ECM deposition and organization during development, wound healing, and pathological diseases. Despite significant advances in understanding the dynamic relationship between mechanical forces and matrix remodeling, many of the unique mechanisms and associated responses to various physical stimuli remain to be elucidated. Fibrosis is a complex disease predominantly characterized by excessive and abnormal fibrous ECM deposition that leads to the failure of various organs: lung, liver, kidney and skin. During the normal wound healing process, injured tissue progresses through phases of hemostasis, acute inflammation, granulation tissue/fibroproliferative, matrix formation, and remodeling. Collectively, the fibro-proliferative stage terminates with the restoration of ECM homeostasis and the disappearance of myofibroblasts, probably through apoptosis. However, the chronic presence of diverse injuries, commonly involving the abnormal persistence of several profibrotic cytokines results in sustained myofibroblast activation, excessive ECM deposition, scar formation, and organ failure. Specifically, transforming growth factor-β (TGF-β) is a master switch that activates critical downstream molecules in the progression of fibrotic disease. Although various strategies designed to interfere with TGF-β expression, receptor binding, and signal transduction have been studied, a clinically safe and effective therapy has not yet been developed. The superficial layer of the lamina propria (SLLP) in the human vocal folds experiences a unique mechanical microenvironment of high frequency vibration during voice production. The presence of macrophages/myofibroblasts in the SLLP of healthy patients suggests that the mechanical stresses imposed during routine speech result in repetitive microtrauma, which is generally repaired without permanent alterations in vocal fold matrix composition or vocal quality. In addition, mechanical forces have recently been shown to alter the fibrotic phenotype in fibrotic fibroblasts. Therefore, the objective of this research is to understand the mechanisms regulating fibroblast matrix metabolism in the SLLP and investigate the potential of vibratory stimulation for treatment of fibrotic diseases. First, we characterized the transcriptional and translational changes of human dermal fibroblasts in response to vibratory stimulation and demonstrated that vibratory stimulation led to the down-regulation of the TGF-β signaling through reduced expression of TGF-β receptors and Smad signal transduction molecules and increased expression of SMAD7, ubiquitin ligases, and SIK1 and SKIL, transcriptional repressors responsible for signaling inhibition. Second, we then investigated the effects of variable vibratory regimes defined by varying frequency, amplitude, and duration on the expression of ECM-related transcripts in human dermal fibroblast and found significant dose-dependent and temporal changes in mRNA expression levels of HA-related molecules and profibrotic cytokines, while type I and III collagen expression was consistently down-regulated across a broad range of parameters. Finally, we tested the potential therapeutic efficacy of vibration for reversing the fibrotic phenotype in scleroderma-derived fibroblasts. These studies showed that vibratory stimulation significantly reduced the mRNA levels of sclerotic pathogenic targets and collagen synthesis and accumulation. These studies, therefore, suggest that vibration can be used as a clinical mechanotherapy for a wide range of fibrotic diseases such as systemic sclerosis and idiopathic pulmonary fibrosis.

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