Indiana University-Purdue University Indianapolis (IUPUI) / Our ability to hear relies on sensory cells found in the inner ear that transduce sound into biological signals. Microvilli-like protrusions called stereocilia are bundled on the apical surfaces of these cells and allow them to respond to sound-evoked vibrations. The architecture of the stereocilia bundle is highly patterned to ensure normal hearing. Filaments of polymerized actin proteins are bundled in parallel into large cylindrical structures that define the dimensions of stereocilia. This network is then anchored to the cell by inserting into another actin-based structure called the cuticular plate, which forms a gel-like structure and facilitates the mechanical properties of the bundle. The shape of the bundle is determined through tissue-level and intrinsic polarization signaling pathways. Auditory brainstem-evoked response testing, immunofluorescence imaging, scanning electron microscopy, and biochemical labeling techniques were used to study how the ADF/cofilin family of actin filament severing and depolymerizing proteins contributes to the development of the stereocilia bundle. Loss of these proteins disrupts the normal bundle patterning process, changes the lengths and widths of stereocilia, and alters the regulation of filament ends near the ion channel at stereocilia tips that is responsible for mechanotransduction. The activity of this channel regulates ADF/cofilins and the actin at stereocilia tips. Aberrant actin growth in actin networks beneath the stereocilia bundle influences the bundle patterning process, causes dysmorphic bundles to form. This work identifies that ADF/cofilins are necessary during auditory sensory cell development to facilitate normal bundle patterning and establishes this protein family as a molecular link between mechanotransduction and stereocilia bundle maturation.

Indiana University-Purdue University Indianapolis (IUPUI) / Bone loss diseases, including osteoporosis affect millions of people worldwide. Understanding the underlying mechanisms behind bone homeostasis and adaptation is essential to uncovering new therapeutic targets for the prevention and treatment of bone loss diseases. Primary cilia have been implicated in the development and mechanosensation of various tissue types, including bone. The goal of the studies outlined in this thesis is to determine the mechanosensory role of primary cilia in bone cell function, bone growth, and adaptation. This goal was achieved by exploring two specific scenarios. In the first study, mice models with conditional knockouts of MKS5, a ciliary protein, in osteocytes were utilized to demonstrate that dysfunctional primary cilia in those cells result in impaired loading-induced bone formation. The hypothesis tested is that the existence of functioning primary cilia on osteocytes is crucial for proper bone adaptation following stress. The results of this study support the hypothesis, with the conditional knockout mice showing significantly lower loading-induced bone formation compared to controls. The second study highlighted the importance of the osteoblast primary cilia in bone growth by using mice models with osteoblast-specific deletion of the cilia. The hypothesis tested is that the presence of the primary cilia is crucial for proper bone growth. The results show that conditional knockout mice have lower body weights, decreased femur length, and a significantly lower rate of bone formation, confirming that the primary cilia play a great role in bone growth and development. This study has highlighted the role of primary cilia in bone health and this topic merits further investigation.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20587号 / 農博第2239号 / 新制||農||1052(附属図書館) / 学位論文||H29||N5076(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 和光, 教授 矢﨑 一史, 教授 宮川 恒 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Due to mechanical loading, cartilage experiences distortional change, volumetric change, and fluid flow. Research has shown cells to be responsive to unconfined compression, a load that produces all three conditions. To isolate the factor(s) responsible for chondrogenesis, the first goal of this research was to design and implement a device for the application of shear deformation to cells. Secondly, using this device, Stage 23/24 chick limb bud cells were suspended in 2% alginate and subjected to 20% shear deformation at 1 Hz. for two hours daily for three days. Gene expression, DNA content, sGAG content, and cartilage nodule formation were determined after eight days in culture and compared to results obtained for non-loaded cells. Results indicated that shear deformation at the applied level did not have a significant effect on chondrogenesis in Stage 23/24 chick limb bud cells, suggesting that this cell type is not extremely sensitive to distortional change.

The global objective of this research was to identify the proteins secreted by stem cells in response to mechanical stress. Since it has been shown in previous studies that conditioned medium from compressed chick limb bud cells cultured in alginate can initiate chondrogenesis in non-compressed cells, it was hypothesized that the conditioned medium contains valuable growth/differentiation factors. Due to cartilage?s limited capacity for repair, factors that stimulate stem-cell mediated regeneration are highly sought. To discern these proteins, conditioned medium was collected from cyclically compressed stage 23/24 chick limb buds suspended in alginate. The proteins were extracted, separated by 2-D gel electrophoresis, and evaluated by mass spectroscopy. While a few regulators of chondrogenesis were observed, such as FGF receptor, actin, and IP3 receptor, many potential peptides were not found in the database. However, this study showed that ascertaining proteins produced by chondrocytes in response to mechanical stimulation should be pursued.

No description available.

Actin stress fibers (SFs) are mechanosensitive structural elements that respond to applied strain to regulate cell morphology, signal transduction, and cell function. The purpose of this dissertation is to elucidate the effects of mechanical stretch on cell mechanobiology via the following three aims. First, a sarcomeric model of SFs was developed to describe the role of actomyosin crossbridge cycling in SF tension regulation and reorientation in response to various modes of stretch. Using model parameters extracted from literature, this model described the dependence of cyclic stretch-induced SF alignment on a two-dimensional (2-D) surface on positive perturbations in SF tension caused by the rate of lengthening, which was consistent with experimental findings. Second, the sarcomeric model was used to predict how stretch-induced pro-inflammatory mechanotransduction depends on the mode of strain application. Together with experimental data, the results indicated that stretch-induced stress fiber alignment, MAPK activations and downstream pro-inflammatory gene expressions are dependent on SF strain rate (and related changes in SF tension) rather than SF turnover. Third, to produce biocompatible materials that are both mechanically resilient under (physiological) load and also mechanosensitive, a novel hybrid engineered tissue was developed that transmits strain stimuli to cells residing in three-dimensional (3-D) collagen microspheres. However, the macroscopic stress is largely borne by a more resilient acellular polyethylene glycol diacrylate (PEGDA) hydrogel supporting the microspheres. Careful analysis indicated that cell alignment occurs prior to significant collagen fibril alignment.

Hypercholesterolemia is a significant risk factor in the development of cardiovascular disease and is associated with chronic leukocyte adhesion in the microvasculature. While the underlying mechanisms behind this have yet to be determined, it may be possible that hypercholesterolemia impairs the fluid shear stress (FSS) inactivation of neutrophils through the rigidifying effect of cholesterol on membrane fluidity. FSS restricts surface expression of CD18 integrins through cathepsin B (ctsB) proteolysis, which minimizes neutrophil adhesivity. If hypercholesterolemia blocks FSS mechanotransduction, then the inhibition of CD18 cleavage may link pathologic blood cholesterol elevations with dysregulated neutrophil adhesion. We hypothesized that elevated cholesterol contributes to dysregulated neutrophil adhesion by impairing ctsB FSS-induced CD18 cleavage through membrane fluidity changes. In the first part of this study, we demonstrated that FSS-induced CD18 cleavage is a robust response of neutrophils and involves selective cleavage of macrophage 1-antigen (Mac1) through ctsB proteolysis. The second part of this study confirmed that ctsB regulates neutrophil adhesion through its proteolytic actions on Mac1, an important integrin involved in adhesion and chemotaxis. Specifically, ctsB accelerated neutrophil motility through an effect on Mac1 integrins during pseudopod retraction. Furthermore, by using a flow-based assay to quantify the mechanoregulation of neutrophil adhesivity, we demonstrated that FSS-induced ctsB release promoted neutrophil detachment from platelet-coated substrates and unstimulated endothelium. For the third part of this study, we linked cholesterol-related membrane fluidity changes with the ability of FSS to restrict neutrophil adhesion through Mac1. We also determined that pathologic cholesterol elevations associated with hypercholesterolemia could block FSS-induced Mac1 cleavage and were linked to disrupted tissue blood flow. This was accomplished using low-density lipoprotein receptor deficient (LDLR-/-) mice fed a high-fat diet. Ultimately, the results provided in the present study confirmed that cholesterol-related changes in membrane fluidity blocked the ability of ctsB to regulate neutrophil adhesion through FSS-induced Mac1 cleavage. This implicates an impaired neutrophil FSS mechanotransduction response in the dysregulation of neutrophil adhesion associated with hypercholesterolemia. Since dysregulated adhesion may be one of the earliest upstream features of cardiovascular disease associated with hypercholesterolemia, the present study provides a foundation for identifying a new mechanobiological factor in the pathobiology of microcirculatory dysfunction.

Chapter 1 of this work focuses on alternative splicing of titin as a proof of concept therapy for treating diastolic dysfunction and restrictive filling in a genetic murine model (Ttn^(ΔIAjxn)). The Ttn^(ΔIAjxn) mouse has increased strain on the spring region of titin and acts as a mechanical analogue of the titin-based increase in passive myocardial stiffness found in patients with heart failure and preserved ejection fraction (HFpEF). HFpEF is a complex disease characterized by diastolic dysfunction, exercise intolerance, and concentric hypertrophic remodeling. Approximately half all of heart failure patients suffer from diastolic dysfunction, however, no effective therapy exists for treating this pervasive syndrome. Titin, the largest known protein and molecular spring in the heart, has emerged as a prime candidate for therapeutic targets aimed at restoring compliance to the sarcomere in order to improve diastolic function. Titin has two main cardiac isoforms that are regulated by alternative splicing; the smaller N2B isoform (~3.0 MDa) and the larger more compliant N2BA isoform (~3.3 MDa). Diastolic stiffness of the left ventricle is dependent upon the N2BA:N2B isoform ratio. In the first half of this work, we modified these two primary isoforms by inhibiting the known titin splicing factor Rbm20. We demonstrate that Rbm20 reduction restores diastolic function, improves exercise tolerance and attenuates afterload induced pathologic remodeling of the left ventricle in Ttn^(ΔIAjxn) mice.The work in chapter 2 is focused on studies using the previously published N2B knock out (KO) murine model. The N2B spring element found in cardiac titin's I-band region has been proposed as a sensor and signaling "hot spot" in the sarcomere. This study investigates the role of titin's cardiac specific N2B element as a mechano-sensor for stress and strain induced remodeling of the heart. The N2B KO mouse was subjected to a variety of stressors including transverse aortic constriction (TAC), aortocaval fistula (ACF), chronic swimming, voluntary running and isoproterenol stimulation. Our data revealed that the N2B element is essential in preload stimulated cardiac hypertrophy as well as remodeling due to beta-adrenergic stress. Cardiac hypertrophy is a common maladaptive feature of heart failure patients and the mechanical triggers that determine pathologic growth are not well understood. My work in the N2B KO mouse reveal titin's important role in cardiac remodeling.

Bladder obstruction (neurogenic or anatomic) induces strain injury in detrusor smooth muscle cells. Signaling via strain injury in other systems has been highly studied, while in bladder obstruction, it has been quite limited to a small number of pathways. In our study we have examined the effects of strain injury using a combination of in vivo, ex vivo and in vitro models, with the aim of understanding disease pathogenesis in the bladder. Using a combination of literature searches, phospho-protein screens and pathway analysis, we uncovered three pathways activated by mechanical strain, ERK, STAT3 and mTOR, with potential for changing not only the way we understand but also the way we treat obstructive myopathies of the bladder. We found that not only were these pathways activated in response to strain and distension injury of BSMC, but they were also responsible for proliferation and sometimes de-differentiation. Included herein are three chapters, published in 2006 and 2010, on the role of ERK, STAT3 and mTOR pathways in bladder smooth muscle cell proliferation and differentiation, 8 Appendices containing the first pages of other papers and reviews published during the course of my studies.