Biomedical subjects

Hiroki Yokota

Publications and source records attributed to Hiroki Yokota.

14 recordsLinked to original sources

PubMed2006

Diaphyseal bone formation in murine tibiae in response to knee loading.

Mechanical stimulation is critical for bone architecture and bone mass. The aim of this study was to examine the effects of mechanical loads applied to the knee. The specific question was whether loads applied to the tibial epiphysis would enhance bone formation in the tibial diaphysis. In C57/BL/6 mice, loads of 0.5 N were applied for 3 min per day for 3 days at 5, 10, or 15 Hz. Bone samples were harvested 13 days after the last loading. The strains were measured 13 +/- 2 microstrains at 5 Hz in the diaphysis. The histomorphometric data in the diaphysis clearly showed enhanced bone formation. First, compared with nonloaded control the cross-sectional cortical area was increased by 11% at 5 Hz and 8% at 10 Hz (both P < 0.05). Second, the cortical thickness was elevated by 12% at 5 Hz (P < 0.01) and 8% at 10 Hz (P < 0.05). Third, mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS) were increased at 5 Hz (P < 0.01 for MS/BS; P < 0.001 for MAR and BFR/BS) and at 10 Hz (P < 0.05 for MS/BS; P < 0.01 for MAR and BFR/BS). Bone formation was enhanced more extensively in the medial side than the lateral or the posterior side. The results reveal that knee loading is an effective means to enhance bone formation in the tibial diaphysis in a loading-frequency dependent manner without inducing significant in situ strain at the site of bone formation.

Adaptation, Physiological↗

PubMed2005

Principal component analysis for predicting transcription-factor binding motifs from array-derived data.

BACKGROUND: The responses to interleukin 1 (IL-1) in human chondrocytes constitute a complex regulatory mechanism, where multiple transcription factors interact combinatorially to transcription-factor binding motifs (TFBMs). In order to select a critical set of TFBMs from genomic DNA information and an array-derived data, an efficient algorithm to solve a combinatorial optimization problem is required. Although computational approaches based on evolutionary algorithms are commonly employed, an analytical algorithm would be useful to predict TFBMs at nearly no computational cost and evaluate varying modelling conditions. Singular value decomposition (SVD) is a powerful method to derive primary components of a given matrix. Applying SVD to a promoter matrix defined from regulatory DNA sequences, we derived a novel method to predict the critical set of TFBMs. RESULTS: The promoter matrix was defined to establish a quantitative relationship between the IL-1-driven mRNA alteration and genomic DNA sequences of the IL-1 responsive genes. The matrix was decomposed with SVD, and the effects of 8 potential TFBMs (5'-CAGGC-3', 5'-CGCCC-3', 5'-CCGCC-3', 5'-ATGGG-3', 5'-GGGAA-3', 5'-CGTCC-3', 5'-AAAGG-3', and 5'-ACCCA-3') were predicted from a pool of 512 random DNA sequences. The prediction included matches to the core binding motifs of biologically known TFBMs such as AP2, SP1, EGR1, KROX, GC-BOX, ABI4, ETF, E2F, SRF, STAT, IK-1, PPARgamma, STAF, ROAZ, and NFkappaB, and their significance was evaluated numerically using Monte Carlo simulation and genetic algorithm. CONCLUSION: The described SVD-based prediction is an analytical method to provide a set of potential TFBMs involved in transcriptional regulation. The results would be useful to evaluate analytically a contribution of individual DNA sequences.

Algorithms↗

PubMed2005

Osteoblast responses one hour after load-induced fluid flow in a three-dimensional porous matrix.

When bone is loaded, substrate strain is generated by the external force and this strain induces fluid flow that creates fluid shear stress on bone cells. Our current understanding of load-driven gene regulation of osteoblasts is based primarily on in vitro studies on planer two-dimensional tissue culture substrates. However, differences between a flat layer of cells and cells in 3-dimensional (3D) ECM are being recognized for signal transduction. Proliferation and differentiation of osteoblasts are affected by substrate geometry. Here we developed a novel 3D culture system that would mimic physiologically relevant substrate strain as well as strain-induced fluid flow in a 3D porous collagen matrix. The system allowed us to evaluate the responses of osteoblasts in a 3D stress-strain environment similar to a mechanical field to which bone is exposed. Using MC3T3-E1 osteoblasts grown in the 3D collagen matrix with and without hydroxyapatite deposition, we tested the role of strain and the strain-induced fluid flow in the expression of the load-responsive genes such as c-fos, egr1, cox2, osteopontin, and mmp1B involved in transcriptional regulation, osteogenesis, and rearrangement of ECM. Strain-induced fluid flow was visualized with a microspheres approximately 3 microm in diameter in real time, and three viscoelastic parameters were determined. The results obtained by semi-quantitative PCR, immunoblot assay, enzymatic activity assays for collagenase and gelatinase, and mechanical characterization of collagen matrices supported the dominant role of strain-induced fluid flow in expression of the selected genes one hour after the mechanical treatment.

3T3 Cells↗

PubMed2005

Osteogenic potentials with joint-loading modality.

Osteogenic potentials with a novel joint-loading modality were examined, using mouse ulnae as a model system. Load-induced deformation of rigid bone is known to generate interstitial fluid flow and stimulate osteogenesis. However, in most of the previous studies, loads were applied to cortical bone. In the current study, we addressed the question of whether deformation of the epiphysis underneath the joint would enhance bone formation in the epiphysis and the diaphysis. In order to answer the question, we applied lateral loads to a mouse elbow and conducted a bone histomorphometric analysis, as well as measurements of strains and streaming potentials. Compared to the no-loading control, the histomorphometric results showed that 0.5-N loads, applied to the elbow at 2 Hz for 3 min/day for 3 consecutive days, increased the mineralizing surface (two- to threefold), the rate of mineral apposition (three- to fivefold), and the rate of bone formation (six- to eightfold) in the ulna. Strain measurements indicated that strains of around 30 microstrain, induced with the joint-loading modality, were under the minimum effective strain of around 1000 microstrain, which is considered necessary to achieve strain-driven bone formation. To evaluate the induction of fluid flow with the joint-loading modality, streaming potentials were measured in separate experiments, using mouse femurs ex vivo. We found that the streaming potentials correlated to the magnitude of the load applied to the epiphysis (r(2) = 0.92), as well as the flow speed in the medullary cavity (r(2) = 0.93). Taken together, the findings of the current study support the idea of joint-loading driven osteogenesis, through a mechanism that involves the induction of fluid flow in cortical bone.

Animals↗

PubMed2004

Oscillating Couette flow for in vitro cell loading.

Synovial joints are loaded by weight bearing, stretching, and fluid-driven shear. To simulate in vitro fluid-driven shear, we developed an "oscillating Couette flow mechanical shear loader". Oscillating Couette flow mimics relative motion of articular surfaces; hence, characterizing flow-induced shear by the loader enhances understanding of mechanotransduction in the joint tissue. Here, the analytical and computational models for an oscillating Couette flow were used to predict time-varying shear distribution on a plate surface, applying numerical simulation to evaluate the effects of finite plate dimension in a 2D flow. Shear stress on the plate was significantly different from that in simpler models (unbounded plates and viscous low-frequency flow). High-stress spots appeared near the leading and trailing edges of a moving plate, and a relatively uniform shear region was restricted to the interior area. Stress prediction in an example experimental geometry is presented, where the frequency and finite width effects are feasibly accounted.

Cells, Cultured↗

PubMed2004

Bone formation induced by a novel form of mechanical loading on joint tissue.

Because of insufficient mechanical loading, exposure to weightlessness in space flight reduces bone mass. In order to maintain bone mass in a weightless condition, we investigated a novel form of mechanical loading--joint loading. Since some part of gravity-induced loading to our skeletal system is absorbed by viscoelastic deformation of joint tissues, we hypothesized that deformation of joint tissues would generate fluid flow in bone and stimulate bone formation in diaphyseal cortical bone. In order to test the hypothesis, we applied directly oscillatory loading to an elbow joint of mice and conducted bone histomorphometry on the diaphysis of ulnae. Using murine femurs ex vivo, streaming potentials were measured to evaluate a fluid flow induced by joint loading. Bone histomorphometry revealed that compared to no loading control, elbow loading increased mineralizing surface, mineral apposition rate, and bone formation rate 3.2-fold, 3.0-fold, and 7.9-fold, respectively. We demonstrated that joint loading generated a streaming potential in a medullar cavity of femurs. The results support a novel mechanism, in which joint loading stimulates effectively bone formation possibly by generating fluid flow, and suggest that a supportive attachment to joints, driven passively or actively, would be useful to maintain bone mass of astronauts during an exposure to weightlessness.

Animals↗

PubMed2003

CITED2-mediated regulation of MMP-1 and MMP-13 in human chondrocytes under flow shear.

CITED2 (CBP/p300-interacting transactivator with ED-rich tail 2) is a member of the Cited family of nuclear regulators, previously known as mrg1 (melanocyte-specific gene-related gene 1). CITED2 is inducible by varying stimuli including lipopolysaccharide, hypoxia, and cytokines such as interleukin 9 and interferon gamma. Using the immortalized human chondrocyte cell line, C-28/I2, we investigated whether CITED2 could be responsive to mechanical stimuli, and if so, whether CITED2 could mediate shear-driven regulation of matrix metalloproteinase (MMP) genes. The C-28/I2 cells were cultured under flow shear at 1-20 dyn/cm2, and the role of CIT-ED2 in regulation of MMPs was examined using the plasmids encoding sense and antisense CITED2 DNA sequences. The results showed that flow shear at 5 dyn/cm2 increased CITED2 mRNA and protein levels and down-regulated MMP-1 and MMP-13 mRNA and protein levels as well as enzyme activities. Consistent with the coordinated expression patterns of CITED2 and MMPs, overexpression of CITED2 repressed MMP-1 and MMP-13 mRNA levels and activities, whereas antisense CITED2 plasmids prevented the shear-induced down-regulation of MMP expression. Interleukin-1beta induced the formation of p300-Ets-1 complexes without affecting expression of CITED2. Transforming growth factor-beta as well as flow shear at 5 dyn/cm2 stimulated not only the expression of CITED2 but also the association of CIT-ED2 with p300 by dissociating Ets-1 from p300. These results indicate that CITED2 plays a major role in shear-induced down-regulation of MMP-1 and MMP-13 via a transforming growth factor-beta-dependent pathway.

Cell Line↗

PubMed2003

Effects of broad frequency vibration on cultured osteoblasts.

Bone is subjected in vivo to both high amplitude, low frequency strain, incurred by locomotion, and to low amplitude, broad frequency strain. The biological effects of low amplitude, broad frequency strain are poorly understood. To evaluate the effects of low amplitude strains ranging in frequency from 0 to 50 Hz on osteoblastic function, we seeded MC3T3-E1 cells into collagen gels and applied the following loading protocols for 3 min per day for either 3 or 7 days: (1) sinusoidal strain at 3 Hz, with 0-3000 microstrain peak-to-peak followed by 0.33 s resting time, (2) "broad frequency vibration" of low amplitude strain (standard deviation of 300 microstrain) including frequency components from 0 to 50 Hz, and (3) sinusoidal strain combined with broad frequency vibration (S + V). The cells were harvested on day 4 or 8. We found that the S + V stimulation significantly repressed cell proliferation by day 8. Osteocalcin mRNA was up-regulated 2.6-fold after 7 days of S + V stimulation, and MMP-9 mRNA was elevated 1.3-fold after 3 days of vibration alone. Sinusoidal stimulation alone did not affect the cell responses. No differences due to loading were observed in alkaline phosphatase activity and in mRNA levels of type I collagen, osteopontin, connexin 43, MMPs-1A, -3, -13. These results suggest that osteoblasts are more sensitive to low amplitude, broad frequency strain, and this kind of strain could sensitize osteoblasts to high amplitude, low frequency strain. This suggestion implies a potential contribution of stochastic resonance to the mechanical sensitivity of osteoblasts.

3T3 Cells↗

PubMed2003

Model-based analysis of matrix metalloproteinase expression under mechanical shear.

We report the development of a model-based analysis for identification of the role of transcription factor (TF) binding motifs. A nonlinear mathematical model was formulated to establish the quantitative relationship between the temporal expression profiles and the distribution of known TF binding motifs on regulatory DNA regions. In order to evaluate biological meaning of the nonlinear model, the role of TF binding motifs predicted by the model was examined by a promoter competition assay where specific TF binding motifs were inactivated by a transient transfer of the DNA fragments consisting of the TF binding motifs. Using the shear stress responses of a family of matrix metalloproteinases in human synovial cells as a model system, we showed that the nonlinear formulation was able to approximate the experimentally observed expression profile better than the linear formulation, and the stimulatory and inhibitory roles of TF binding motifs extracted from the model were validated by the competition assay. The results support that an integrated usage of the nonlinear model and the biochemical evaluation assay would contribute to identifying critical regulatory DNA elements in mechanical responses in connective tissue.

Amino Acid Motifs↗

PubMed2003

Expression and activities of matrix metalloproteinases under oscillatory shear in IL-1-stimulated synovial cells.

Matrix metalloproteinases (MMPs) are known to play a critical role in tissue disintegration, and an elevated level of MMPs is observed in synovium and synovial fluid of joints with rheumatoid arthritis. During joint movement, synovial tissue receives various mechanical stimuli, but effects of mechanical challenges on regulation of MMPs in rheumatic synovium are poorly understood. Focusing on cellular responses to oscillatory fluid shear in human synovial cells, we determined the expression of MMP-1 and MMP-13 by polymerase chain reaction and immunoblotting as well as proteolytic activities of total MMPs by a fibril degradation assay and zymography. The results revealed that approximately 0.5 dyn/cm2 oscillatory shear at 1 Hz not only reduced an mRNA level and a protein level of MMP-1 and MMP-13, but it also decreased collagenase and gelatinase activities of total MMPs. Furthermore, the induction of the MMP expression and activities by interleukin-1 was suppressed by the oscillatory shear. Interestingly, the oscillatory shear upregulated the mRNA expression of TIMP-1 and TIMP-2. Our results support a potential role of oscillatory shear in regulating expression and activities of MMPs in the presence and the absence of proinflammatory cytokine.

Arthritis, Rheumatoid↗

PubMed2002

Involvement of the calcium-independent receptor for alpha-latrotoxin in brain ischemia.

Cerebral ischemia is caused by a reduced blood supply to neurons, and vulnerability to neurodegeneration varies considerably among neuronal types. In hippocampus, neurons in the CA1 region are more susceptible to ischemia-induced neuronal death than neurons in the CA3 region, and in response to transient forebrain ischemia a family of calcium-dependent receptors for alpha-latrotoxin is differentially expressed in the two regions. Here, we report that an ischemic insult up-regulated a family of calcium-independent receptors for alpha-latrotoxin (CIRL) mRNAs in CA1 neurons and down-regulated their mRNAs in CA3 neurons. Furthermore, antisense oligonucleotides complementary to CIRL-1 mRNA or CIRL-3 mRNA suppressed neuronal death associated with hypoxia in hippocampal and cortical cell cultures. The observed region-specific CIRL mRNA expression in hippocampus and an in vitro rescue experiment by antisense oligonucleotides against CIRL mRNAs suggest a functional importance of CIRL in neurodegeneration.

Animals↗

PubMed2002

Promoter competition assay for analyzing gene regulation in joint tissue engineering.

We describe a new biochemical technique, "promoter competition assay," for examining the role of cis-acting DNA elements in tissue cultures. Recent advances in tissue engineering permit the culture of a variety of cells. Many tissues are engineered, however, without an appropriate understanding of molecular machinery that regulates gene expression and cellular growth. For elucidating the role of cis-acting regulatory elements in cellular differentiation and growth, we developed the promoter competition assay. This assay uses a transient transfer into cells of double-stranded DNA fragments consisting of cis-acting regulatory elements. The transferred DNA fragments act as a competitor and titrate the function of their genomic counterparts. Using synovial cells derived from a rheumatoid arthritis patient, we examined a role of NF-kappa B binding sites in the regulation of the expression of matrix metalloproteinase (MMP) genes. The results support a stimulatory role of NF-kappa B in transcriptional regulation of MMP-1 and MMP-13.

Arthritis, Rheumatoid↗

PubMed2002

Systems analysis of matrix metalloproteinase mRNA expression in skeletal tissues.

The availability of human genome sequences provides life scientists and biomedical engineers with a challenging opportunity to develop computational and experimental tools for quantitatively analyzing biological processes. In response to a growing need to integrate experimental mRNA expression data with human genome sequences, we present here a unique analysis named "Promoter-Based Estimation (PROBE)" analysis. The PROBE analysis is "systems analysis" of transcriptional processes using control and estimation theories. A linear model was built in order to estimate the mRNA levels of a group of genes from their regulatory DNA sequences. The model was also used to interpret two independent datasets in skeletal tissues. The results demonstrated that the mRNA levels of a family of matrix metalloproteinases can be modeled from a distribution of cis-acting elements on regulatory DNA sequences. The model accurately predicted a stimulatory role of cis-acting elements such as AP1, NFY, PEA3, and Sp1 as well as an inhibitory role of AP2. These predictions are consistent with biological observations, and a specific assay for testing such predictions is proposed. Although eukaryotic transcription is a complex mechanism, the two examples presented here support the potential use of the described analysis for elucidating the functional significance of DNA regulatory elements.

Algorithms↗

PubMed2002

Reduction of cytokine-induced expression and activity of MMP-1 and MMP-13 by mechanical strain in MH7A rheumatoid synovial cells.

Excessive mechanical load induces harmful outcomes for joint diseases, such as osteoarthritis and rheumatoid arthritis, but physical stimuli at appropriate intensity are essential for growth and maintenance of bone and articular cartilage. Using a fibroblast-like synoviocyte cell line derived from a patient with rheumatoid arthritis, we examined the effects of gentle cyclic strain, focusing on the expression and activity of matrix metalloproteinase-1 (MMP-1) and MMP-13. Synovial cells were cultured on a collagen-coated agar block and exposed to 2% cyclic strain at 6 rev./min for 1 h. Expression of MMP-1 and MMP-13 was assayed using semi-quantitative and real-time PCR, as well as immunoblotting. Their activity was measured using spectrofluorometry and zymography. The results showed that the cyclic strain reduced the mRNA and protein levels of MMP-1 and MMP-13, and that both collagenase and gelatinase activity was decreased under the strain. The reduction in MMP activity by the cyclic strain was not achieved by the transcriptional inhibitor, actinomycin D. In the presence of proinflammatory cytokines, such as IL-1 beta and TNF-alpha, the strain reduced the cytokine-induced expression and activities of MMPs. Interestingly, the strain elevated the mRNA level of tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2. These results support a potential role of mechanical strain in down-regulating the cytokine-mediated proteolytic processes in synoviocytes.

Arthritis, Rheumatoid↗