Mechanotransduction and control of valvular cell phenotype as tools to inform valvular pathophysiology




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Valvular degenerative diseases cause significant morbidity and mortality in developed countries. Valvular interstitial cells (VICs) are responsible for pathogenesis of these diseases. In healthy adult valves, VICs remain as quiescent phenotype. In degenerative valves, they become phenotypically activated like myofibroblast. Controlling VIC activation has important therapeutic and tissue engineering implications. Mechanobiology also plays a crucial role in valvular pathophysiology. Various mechanical forces trigger VIC phenotypical transformations and valvular degenerative diseases. However, valvular mechanotransduction pathways of these forces are largely unknown. Valves have an outer endothelium layer consisting of endothelial cells. Endothelium maintains valvular physiology but function of endothelium in valvular mechanotransduction is unknown. In this dissertation, we studied VIC activation in terms VIC morphological distribution in vitro. We also demonstrated control over VIC activation in vitro via interaction with progenitor subpopulations and via variable substrate stiffness. Lastly, we studied valvular mechanotransduction and the endothelial function in it using high throughput analysis of valvular cell and tissue response to strain and substrate stiffness in vitro.

In our study of VIC phenotypes, we detected six distinct VIC morphologies and their relative abundance in pathophysiological states. Morphologies were associated with VIC activation. This in vitro morphology based VIC phenotype detection is simpler and quicker compared to molecular marker-based techniques. We demonstrated control over VIC phenotype in vitro, inducing VIC deactivation by lowering substrate stiffness. Reversibility in VIC activation has important implications in maintaining quiescence for in vitro research with rare human VICs and for tissue engineering purposes. We also demonstrated control over VIC activation, in vitro, using interaction with two progenitor subpopulations of VICs resembling hematopoietic and mesenchymal stem cells. These subpopulation functions were amplified by increasing their concentrations to >50% in VIC cultures. Hematopoietic stem cell subpopulation induced deactivation in VICs. Mesenchymal subpopulations did not show any effect on VIC activation. This is the first study showing function of a progenitor VIC subpopulation in VIC activation and will augment progenitor VIC research which is at its infancy.

Next, we focused on high throughput proteomic analyses of valvular cell and tissue response to variable substrate stiffness and physiologic cyclic strain. Cytoskeletal, signal transduction, oxidative stress and translation proteins were upregulated on stiff substrate suggesting their role in VIC mechanotransduction. Strain resulted in quiescent state with upregulated mechanoreceptors associated with pro-inflammatory and pro-angiogenic activities. Endothelium removal resulted in upregulation of translation suggesting a protective role of valvular endothelium. These high throughput studies will contribute to identifying cell signaling cascades and mechanotransduction pathways in VICs.

Lastly, we also developed a computational model of diastolic heart left ventricle to determine blood flow and wall stress related changes introduced by medical device ‘Mitraclip’. Despite some limitations, the model can be used in future to simulate other left ventricular diastolic phenomena.

This dissertation improves our understanding and control of VIC phenotype structure and function as well as links molecular, cellular and tissue-level valvular responses to static and dynamic stimuli. Taken together, these results expand the picture of valvular cell mechanotransduction, morphology, phenotypical transformation, signaling networks and tissue maintenance.



Heart valve, Pathophysiology, Cell phenotype, Cell morphology, Progenitor, Stem cell, Mechanobiology, Mechanotransduction