京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13195号 / 論医博第2159号 / 新制||医||1030(附属図書館) / (主査)教授 松田 秀一, 教授 三森 経世, 教授 妻木 範行 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Myocardial infarction (MI) is the leading cause of death among men and women in the United States. Once a person suffers from a MI, the heart wall will undergo a dynamic and time dependent change, as it goes through the inflammatory phase, proliferative phase, and healing phase. During these phases, the necrotic tissue is removed, and the extracellular matrix (ECM) that holds the cardiomyocytes is altered by an increase in type I collagen, which leads to a scar formation in the infarcted area. The goal of this dissertation is to better understand the role of the cardiac ECM biomechanics in heart physiology, pathophysiology (MI), and regeneration. Three Aims were hence pursued. In Aim 1, we investigated cardiac ECM architecture in intact acellular hearts using diffusion tensor-magnetic resonance imaging (DT-MRI); additionally, we characterized the biomechanical and structural properties of cardiac ECM at different anatomical locations of the left ventricle wall. In Aim 2, we characterized the biomechanical and structural properties of scar ECM during the acute to chronic stages of MI using a rat heart model, in order to better understanding the time course changes in scar ECM biomechanics/microenvironment. In Aim 3, we determined if large mammals (pig heart model) have the capability to fully regenerate a resected piece of the heart apex during the neonatal stage, in which cardiac ECM is still in a developmental phase. The hope is to apply the obtained knowledge in cardiac ECM biomechanics to improve the effectiveness and efficiency of treatments, such as stem cell injection, scar tissue repairing, and regenerative intervention.

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Vascular endothelial growth factor (VEGF) is required for the development, growth and survival of blood vessels. Endothelial cell behavior is altered by cell substrate stiffness, suggesting that VEGF activity might also be influenced by cell-substrate mechanics. We studied VEGF binding, internalization, and signaling as a function of substrate stiffness using endothelial cells cultured on fibronectin (fn) linked polyacrylamide gels. Individual cell analysis of VEGF-induced calcium fluxes in endothelial cells on various stiffness extracellular matrices (ECM) revealed heterogeneity in our cell population that would have been lost using population based averaging. Cluster analysis of individual cells identified two key groups of reacting cells- a minor fraction of highly reactive cells and the bulk of the cells with minimal activation. At subsaturating VEGF doses, highly active cells were phenotypically smaller and thinner than the bulk population. Overall, cells on our softest substrates (4 kPa) were most sensitive to VEGF. To better understand the mechanisms underlying the changes in VEGF signaling due to stiffness, we explored how matrix binding of VEGF and tethering of cells to the matrix modulates VEGF processing. VEGF-ECM binding was enhanced with heparin pre-treatment, which exposed a cryptic VEGF binding site in the fn ECM. Cell produced ECM on the softest substrates were least responsive to heparin, but the cells internalized more VEGF and showed enhanced VEGF signaling compared to cells on all other substrates. Inhibiting VEGF-matrix binding with sucrose octasulfate decreased cell-internalization of VEGF in all conditions. β1 integrin, which connects cells to fn, modulated VEGF uptake in a stiffness dependent fashion. β1 protein levels were consistent with stiffness, yet cells on hard surfaces showed greater decreases in VEGF internalization than cells on softer matrices after β1 inhibition. Stiff matrices facilitate the unfolding of fn, which may reduce the binding capacity of β1 integrin. Thus a greater proportion of activated β1 integrin may be sensitive to inhibition in the stiff condition as compared to the soft. Ultimately, through analysis of individual and population-based VEGF-cell responses to stiffness, this study provides insight into how signaling dynamics, cell heterogeneity, and microenvironment influence tissue regeneration and response to injury and disease.

Damage to our tissues occurs daily and must be repaired by the body in a timely manner in order to prevent infection and restore tissue integrity. Many cell types are involved in the healing process, but it is the cells of the stroma that are largely responsible for rebuilding fibrous tissue, which provides structure and support for all other cell types during healing. This dissertation focuses on stromal tissue repair, the rebuilding of fibrous tissue by fibroblasts following injury. Specifically, I focus on 1) models to study wound healing in vitro, and the specific biological processes of healing that each model captures, 2) the response of engineered stromal microtissues to different methods of injury, namely laceration and laser ablation, and the subsequent clearance and rebuilding of the extracellular matrix by fibroblasts, and 3) how different types of stromal cells and extracellular matrix proteins contribute to tissue repair in vitro.