IU Indianapolis ScholarWorks :: Browsing by Author "Yokota, Hiroki, 1955-"

Browsing by Author "Yokota, Hiroki, 1955-"

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Application of quantitative analysis in treatment of osteoporosis and osteoarthritis
(2013-11-08) Chen, Andy Bowei; Yokota, Hiroki, 1955-; Na, Sungsoo; Schild, John H.
As our population ages, treating bone and joint ailments is becoming increasingly important. Both osteoporosis, a bone disease characterized by a decreased density of mineral in bone, and osteoarthritis, a joint disease characterized by the degeneration of cartilage on the ends of bones, are major causes of decreased movement ability and increased pain. To combat these diseases, many treatments are offered, including drugs and exercise, and much biomedical research is being conducted. However, how can we get the most out of the research we perform and the treatment we do have? One approach is through computational analysis and mathematical modeling. In this thesis, quantitative methods of analysis are applied in different ways to two systems: osteoporosis and osteoarthritis. A mouse model simulating osteoporosis is treated with salubrinal and knee loading. The bone and cell data is used to formulate a system of differential equations to model the response of bone to each treatment. Using Particle Swarm Optimization, optimal treatment regimens are found, including a consideration of budgetary constraints. Additionally, an in vitro model of osteoarthritis in chondrocytes receives RNA silencing of Lrp5. Microarray analysis of gene expression is used to further elucidate the mode of regulation of ADAMTS5, an aggrecanase associated with cartilage degradation, by Lrp5, including the development of a mathematical model. The math model of osteoporosis reveals a quick response to salubrinal and a delayed but substantial response to knee loading. Consideration of cost effectiveness showed that as budgetary constraints increased, treatment did not start until later. The quantitative analysis of ADAMTS5 regulation suggested the involvement of IL1B and p38 MAPK. This research demonstrates the application of quantitative methods to further the usefulness of biomedical and biomolecular research into treatment and signaling pathways. Further work using these techniques can help uncover a bigger picture of osteoarthritis's mode of action and ideal treatment regimens for osteoporosis.
Effects of interstitial fluid flow and cell compression in FAK and SRC activities in chondrocytes
(2013-11-08) Cho, Eunhye; Na, Sungsoo; Yokota, Hiroki, 1955-; Li, Jiliang
Articular cartilage is subjected to dynamic mechanical loading during normal daily activities. This complex mechanical loading, including cell deformation and interstitial fluid flow, affects chondrocyte mechano-chemical signaling and subsequent cartilage homeostasis and remodeling. Focal adhesion kinase (FAK) and Src are known to be main mechanotransduction proteins, but little is known about the effect of mechanical loading on FAK and Src under its varying magnitudes and types. In this study, we addressed two questions using C28/I2 chondrocytes subjected to the different types and magnitudes of mechanical loading: Does a magnitude of the mechanical loading affect activities of FAK and Src? Does a type of the mechanical loading also affect their activities? Using fluorescence resonance energy transfer (FRET)-based FAK and Src biosensor in live C28/I2 chondrocytes, we monitored the effects of interstitial fluid flow and combined effects of cell deformation/interstitial fluid flow on FAK and Src activities. The results revealed that both FAK and Src activities in C28/I2 chondrocytes were dependent on the different magnitudes of the applied fluid flow. On the other hand, the type of mechanical loading differently affected FAK and Src activities. Although FAK and Src displayed similar activities in response to interstitial fluid flow only, simultaneous application of cell deformation and interstitial fluid flow induced differential FAK and Src activities possibly due to the additive effects of cell deformation and interstitial fluid flow on Src, but not on FAK. Collectively, the data suggest that the intensities and types of mechanical loading are critical in regulating FAK and Src activities in chondrocytes.
The essential role of Stat3 in bone homeostasis and mechanotransduction
(2014-05) Zhou, Hongkang; Li, Jiliang; Marrs, James; Stocum, David L.; Atkinson, Simon; Aguilar, R. Claudio; Yokota, Hiroki, 1955-
Signal Transducer and Activator of Transcription 3 (Stat3) is a transcription factor expressed in bone and joint cells that include osteoblasts, osteocytes, osteoclasts, and chondrocytes. Stat3 is activated by a variety of cytokines and growth factors, including IL-6/gp130 family cytokines. These cytokines not only regulate the differentiation of osteoblasts and osteoclasts, but also regulate proliferation of chondrocytes through Stat3 activation. In 2007, mutations of Stat3 have been confirmed to cause a rare human immunodeficiency disease – Job syndrome which presents skeletal abnormalities like: reduced bone density (osteopenia), scoliosis, hyperextensibility of joints, and recurrent pathological bone fractures. Changes in the Stat3 gene alter the structure and function of the Stat3 proteins, impairing its ability to control the activity of other genes. However, little is known about the effects of Stat3 mutations on bone cells and tissues. To investigate the in vivo physiological role of Stat3 in bone homeostasis, osteoblast/osteocyte-specific Stat3 knockout (KO) mice were generated via the Cre-LoxP recombination system. The osteoblast/osteocyte-specific Stat3 KO mice showed bone abnormalities and an osteoporotic phenotype because of a reduced bone formation rate. Furthermore, inactivation of Stat3 decreased load-driven bone formation, and the disruption of Stat3 in osteoblasts suppressed load-driven mitochondrial activity, which led to an elevated level of reactive oxygen species (ROS) in cultured primary osteoblasts. Stat3 has been found to be responsive to mechanical stimulation, and might play an important role in mechanical signal transduction in osteocytes. To investigate the role Stat3 plays in mechanical signaling transduction, osteocyte-specific Stat3 knockout (KO) mice were created. Inactivation of Stat3 in osteocytes presented a significantly reduced load-driven bone formation. Decreased osteoblast activity indicated by reduced osteoid surface was also found in osteocyte-specific Stat3 KO mice. Moreover, sclerostin (SOST) protein which is a critical osteocyte-specific inhibitor of bone formation, its encoded gene SOST expression has been found to be enhanced in osteocyte-specific Stat3 KO mice. Thus, these results clearly demonstrated that Stat3 plays an important role in bone homeostasis and mechanotransduction, and Stat3 is not only involved in bone-formation-important genes regulation in the nucleus but also in mediation of ROS and oxidative stress in mitochondria.
Experimental and Computational Analysis of Dynamic Loading for Bone Formation
(2013-11-12) Dodge, Todd Randall; Wallace, Joseph; Na, Sungsoo; Yokota, Hiroki, 1955-; Schild, John H.
Bone is a dynamic tissue that is constantly remodeling to repair damage and strengthen regions exposed to loads during everyday activities. However, certain conditions, including long-term unloading of the skeleton, hormonal imbalances, and aging can disrupt the normal bone remodeling cycle and lead to low bone mass and osteoporosis, increasing risk of fracture. While numerous treatments for low bone mass have been devised, dynamic mechanical loading modalities, such as axial loading of long bones and lateral loading of joints, have recently been examined as potential methods of stimulating bone formation. The effectiveness of mechanical loading in strengthening bone is dependent both on the structural and geometric characteristics of the bone and the properties of the applied load. For instance, curvature in the structure of a bone causes bending and increased strain in response to an axial load, which may contribute to increased bone formation. In addition, frequency of the applied load has been determined to impact the degree of new bone formation; however, the mechanism behind this relationship remains unknown. In this thesis, the application of mechanical loading to treat osteoporotic conditions is examined and two questions are addressed: What role does the structural geometry of bone play in the mechanical damping of forces applied during loading? Does mechanical resonance enhance geometric effects, leading to localized areas of elevated bone formation dependent on loading frequency? Curvature in the structure of bone was hypothesized to enhance its damping ability and lead to increased bone formation through bending. In addition, loading at frequencies near the resonant frequencies of bone was predicted to cause increased bone formation, specifically in areas that experienced high principal strains due to localized displacements during resonant vibration. To test the hypothesis, mechanical loading experiments and simulations using finite element (FE) analysis were conducted to characterize the dynamic properties of bone. Results demonstrate that while surrounding joints contribute to the greatest portion of the damping capacity of the lower limb, bone absorbs a significant amount of energy through curvature-driven bending. In addition, results show that enhanced mechanical responses at loading frequencies near the resonant frequencies of bone may lead to increased bone formation in areas that experience the greatest principal strain during vibration. These findings demonstrate the potential therapeutic effects of mechanical loading in preventing costly osteoporotic fractures, and explore characteristics of bone that may lead to optimization of mechanical loading techniques. Further investigation of biomechanical properties of bone may lead to the prescribing of personalized mechanical loading treatments to treat osteoporotic diseases.
In Vitro and In Silico Analysis of Osteoclastogenesis in Response to Inhibition of De-phosphorylation of EIF2alpha by Salubrinal and Guanabenz
(2013) Tanjung, Nancy Giovanni; Yokota, Hiroki, 1955-; Ji, Julie; Na, Sungsoo; Berbari, Edward J.
An excess of bone resorption over bone formation leads to osteoporosis, resulting in a reduction of bone mass and an increase in the risk of bone fracture. Anabolic and anti-resorptive drugs are currently available for treatment, however, none of these drugs are able to both promote osteoblastogenesis and reduce osteoclastogenesis. This thesis focused on the role of eukaryotic translation initiation factor 2 alpha (eIF2alpha), which regulates efficiency of translational initiation. The elevation of phosphorylated eIF2alpha was reported to stimulate osteoblastogenesis, but its effects on osteoclastogenesis have not been well understood. Using synthetic chemical agents such as salubrinal and guanabenz that are known to inhibit the de-phosphorylation of eIF2alpha, the role of phosphorylation of eIF2alpha in osteoclastogenesis was investigated in this thesis. The questions addressed herein were: Does the elevation of phosphorylated eIF2alpha (p-eIF2alpha) by salubrinal and guanabenz alter osteoclastogenesis? If so, what regulatory mechanism mediates the process? It was hypothesized that p-eIF2alpha could attenuate the development of osteoclast by regulating the transcription factor(s) amd microRNA(s) involved in osteoclastogenesis. To test this hypothesis, we conducted in vitro and in silico analysis of the responses of RAW 264.7 pre-osteoclast cells to salubrinal and guanabenz. First, the in vitro results revealed that the elevated level of phosphorylated eIF2alpha inhibited the proliferation, differentiation, and maturation of RAW264.7 cells and downregulated the expression of NFATc1, a master transcription factor of osteoclastogenesis. Silencing eIF2alpha by RNA interference suppressed the downregulation of NFATc1, suggesting the involvement of eIF2alpha in regulation of NFATc1. Second, the in silico results using genome-wide expression data and custom-made Matlab programs predicted a set of stimulatory and inhibitory regulator genes as well as microRNAs, which were potentially involved in the regulation of NFATc1. RNA interference experiments indicated that the genes such as Zfyve21 and Ddit4 were primary candidates as an inhibitor of NFATc1. In summary, the results showed that the elevation of p-eIF2alpha by salubrinal and guanabenz leads to attenuation of osteoclastogenesis through the downregulation of NFATc1. The regulatory mechanism is mediated by eIF2alpha signaling, but other signaling pathways are likely to be involved. Together with the previous data showing the stimulatory role of p-eIF2alpha in osteoblastogenesis, the results herein suggest that eIF2alpha-mediated signaling could provide a novel therapeutic target for treatment of osteoporosis by promoting bone formation and reducing bone resorption.
Multi-scale analysis of morphology, mechanics, and composition of collagen in murine osteogenesis imperfecta
(2013-11-06) Bart, Zachary Ryan; Wallace, Joseph; Na, Sungsoo; Yokota, Hiroki, 1955-; Schild, John H.
Osteogenesis imperfecta is a rare congenital disease commonly characterized by brittle bones caused by mutations in the genes encoding Type I collagen, the single most abundant protein produced by the body. The murine model (oim) exists as a natural mutation of this protein, converting its heterotrimeric structure of two Col1a1 molecules and a single Col1a2 molecule into homotrimers composed of only the former. This defect impacts bone mechanical integrity, greatly weakening their structure. Femurs from male wild type (WT), heterozygous (oim/+), and homozygous (oim/oim) mice, all at 12 weeks of age, were assessed using assays at multiple length scales with minimal sample processing to ensure a near-physiological state. Atomic force microscopy (AFM) demonstrated detectable differences in the organization of collagen at the nanometer scale that may partially attribute to alterations in material and structural behavior obtained through mechanical testing and reference point indentation (RPI). Changes in geometric and chemical structure through the use of µ-Computed Tomography and Raman spectroscopy respectively indicate a smaller, brittle phenotype caused by oim. Changes within the periodic D-spacing of collagen point towards a reduced mineral nucleation site, supported by reduced mineral crystallinity, resulting in altered material and structural behavior in oim/oim mice. Multi-scale analyses of this nature offer much in assessing how molecular changes can compound to create a degraded, brittle phenotype.
THE ROLE OF DAP-KINASE IN MODULATING VASCULAR ENDOTHELIAL CELL FUNCTION UNDER FLUID SHEAR STRESS
(2010-05-05T15:00:50Z) Rennier, Keith; Ji, Julie Ying Hui; Yokota, Hiroki, 1955-; Li, Jiliang
Atherosclerosis preferentially develops in vascular regions of low or disturbed flow and high spatial gradients. Endothelial cells that line the vessel walls actively participate in translating mechanical stimuli, shear stress due to fluid flow, into intracellular signals to regulate cellular activities. Atherosclerosis is a chronic disease. During its development, a cascade of inflammatory signals alters the arterial endothelial homeostatic functions. Death-associated protein (DAP) kinase and its correlated pathway have been associated with cell apoptosis, turnover, and cytoskeleton remodeling in cellular networks, ultimately leading to changes in cell motility and vascular wall permeability. DAP-kinase is also highly regulated by inflammatory triggers such as TNF-α. This thesis investigates DAP-kinase modulation due to shear stress, and the role of DAP-kinase activity in endothelial responses toward applied shear stress. Using bovine aortic endothelial cells (BAEC), DAP-kinase expression is demonstrated in both sheared (10 dynes/cm2) and static conditions. Overall DAPK expression increased with extended shearing, while the presence of phosphorylated DAPK decreased with applied shear stress, as demonstrated in Western blot analysis. In correlation, DAPK RNA expression profiles were explored to understand pre-translational behavior and to understand just how shear stress influences DAPK expression over time. There is a temporal increase in DAPK mRNA, occurring at earlier time points when compared to DAPK protein expression, displaying the precedence of mRNA expression leading to increased translation into protein. From our apoptosis assay results, shear stress reduces apoptotic and late stage/necrotic cell fractions. The exposure of shear stress potentially plays a role in inhibiting apoptosis activation and TNF-α induced death cascade. Overall, the apoptosis activity influenced by shear further exhibits a possible connection between shear stress and apoptosis inhibition. The shear stress ultimately decreases overall apoptosis, while DAPK expression is increased. Therefore, DAPK may have a function in other possible mechano-transduction cascades, when endothelial cells are exposed to constant shear. Our data suggests shear stress modulation of DAP-kinase expression and activity, and the potential crosstalk of mechano-transduction and DAPK/apoptosis pathway, may lead to further understanding the responsibility of DAPK in endothelial cell function.
Signaling Pathways Involved in Mechanical Stimulation and ECM Geometry in Bone Cells
(2010-07-27T20:24:41Z) Jiang, Chang; Yokota, Hiroki, 1955-; Liu, Yunlong; Ji, Julie
The proliferation and differentiation of osteoblasts are influenced by mechanical and geometrical growth environments. A specific aim of my thesis was the elucidation of signaling pathways involved in mechanical stimulation and geometric alterations of the extracellular matrix (ECM). A pair of questions addressed herein was (a) Does mechanical stimulation modulate translational regulation through the phosphorylation of eukaryotic initiation factor 2 (eIF2)? (b) Do geometric alterations affect the phosphorylation patterns of mitogen-activated protein kinase (MAPK) signaling? My hypothesis was mechanical stress enhances the proliferation and survival of osteoblasts through the reduction in phosphorylation of eIF2, while 3-dimensional (3D) ECM stimulates differentiation of osteoblasts through the elevation of phosphorylation of p38 MAPK. First, mechanical stimulation reduced the phosphorylation of eIF2. Furthermore, flow pre-treatment reduced thapsigargin-induced cell mortality through suppression of phosphorylation of protein kinase RNA-like ER kinase (Perk). However, H2O2-driven cell mortality, which is not mediated by Perk, was not suppressed by mechanical stimulation. Second, in the ECM geometry study, the expression of the active (phosphorylated) form of p130Cas, focal adhesion kinase (FAK) and extracellular signal-regulated protein kinase (ERK) was reduced in cells grown in the 3D matrix. Conversely, phosphorylation of p38 MAPK was elevated in the 3D matrix and its up-regulation was linked to an increase in mRNA levels of dentin matrix protein 1 and bone sialoprotein. In summary, our observations suggest the pro-survival role of mechanical stimulation and the modulation of osteoblastic fates by ECM geometry.

Browsing by Author "Yokota, Hiroki, 1955-"

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