Language：English   Publishing type：Research paper (scientific journal)  

Cardiomyocytes are contractile cells that regulate heart contraction. Ca2+ flux via Ca2+ channels activates actomyosin interactions, leading to cardiomyocyte contraction, which is modulated by physical factors (e.g., stretch, shear stress, and hydrostatic pressure). We evaluated the mechanism triggering slow contractions using a high-pressure microscope to characterize changes in cell morphology and intracellular Ca2+ concentration ([Ca2+]i) in mouse cardiomyocytes exposed to high hydrostatic pressures. We found that cardiomyocytes contracted slowly without an acute transient increase in [Ca2+]i, while a myosin ATPase inhibitor interrupted pressure-induced slow contractions. Furthermore, transmission electron microscopy showed that, although the sarcomere length was shortened upon the application of 20 MPa, this pressure did not collapse cellular structures such as the sarcolemma and sarcomeres. Our results suggest that pressure-induced slow contractions in cardiomyocytes are driven by the activation of actomyosin interactions without an acute transient increase in [Ca2+]i.

DOI： 10.1016/j.bpj.2022.07.016

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

Gravity determines shape of body tissue and affects the functions of life, both in plants and animals. The cellular response to gravity is an active process of mechanotransduction. Although plants and animals share some common mechanisms of gravity sensing in spite of their distant phylogenetic origin, each species has its own mechanism to sense and respond to gravity. In this review, we discuss current understanding regarding the mechanisms of cellular gravity sensing in plants and animals. Understanding gravisensing also contributes to life on Earth, e.g., understanding osteoporosis and muscle atrophy. Furthermore, in the current age of Mars exploration, understanding cellular responses to gravity will form the foundation of living in space.

DOI： 10.1038/s41526-020-00130-8

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Other Link： http://www.nature.com/articles/s41526-020-00130-8

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

Abstract

Background

The use of animal models in cardiovascular research is associated with two serious, intrinsic problems: inaccuracy in the extrapolation of data obtained from animals such as rodents due to different cardiac physiology and animal ethics.

Purpose

To develop an artificial human heart model for cardiovascular research using organ-on-a-chip technology and human cells

Methods

Organ chips made of silicone (polydimethylsiloxane) that have two microfluidic channels, a top channel and a bottom channel, separated by a 50-μm thick membrane with 7-μm pores hexagonally packed at 40-μm intervals (Figure 1), were used in this study.

We seeded 10,000 human umbilical vein endothelial cells on the membrane surface in the bottom channel to mimic the vasculature. Next, we seeded a mixture of 100,000 human-induced pluripotent stem cells (hiPSCs) and 50,000 human gingival fibroblasts on the membrane surface in the top channel (Figure 1). Human gingival fibroblasts facilitate cardiac differentiation of hiPSCs [1]. We performed the cardiac differentiation of hiPSCs using a previously described protocol [1]. Culture medium was perfused at a constant rate of 60 μl/h to maintain the culture.

Results

We observed spontaneous contraction of hiPSC-derived cardiomyocytes 20–26 days after the start of the differentiation protocol. Simultaneously, we conducted live intracellular calcium imaging using a fluorogenic calcium-sensitive dye, Cal-520 AM (5 μM in the culture medium). hiPSC-derived cardiomyocytes exhibited a periodic, coordinated pattern of calcium influx synchronised with their contraction under fluorescence microscopy (Figure 2A). Moreover, we found that the β-adrenergic agonist noradrenaline elevated the heart rate of hiPSC-derived cardiomyocytes on the organ chips in a dose-dependent manner (Figures 2A, B).

After observing the contraction and intracellular calcium influx of hiPSC-derived cardiomyocytes, we performed immunocytochemistry. Confocal microscopy indicated that the fluorescent signal obtained from anti-cardiac troponin T antibody staining in the top channel exhibited a typical striated pattern with 1.56±0.12-μm interval that reflected sarcomere structure (Figure 2C, yellow). Moreover, the fluorescent signal obtained from anti-CD31 antibody staining in the bottom channel exhibited a typical pattern at the boundary between cells, which is expected at the cell–cell junction of endothelial cells.

Conclusion

We developed a human heart-on-a-chip model that was confirmed by the functional response to noradrenaline and the histological evidence of sarcomere structure and vasculature, with a capability of live imaging. We expect that this model would be useful for examining the physiological function and for the pharmacological analysis of not only the normal heart but also the heart that reflects specific patient's pathophysiology using patient-derived hiPSCs.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Japan Society for the Promotion of Science Figure 1Figure 2

DOI： 10.1093/eurheartj/ehab724.3190

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Authorship：Last author   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

Gravity affects the function and maintenance of organs, such as bones, muscles, and the heart. Several studies have used DNA microarrays to identify genes with altered expressions in response to gravity. However, it is technically challenging to combine the results from various microarray datasets because of their different data structures. We hypothesized that it is possible to identify common changes in gene expression from the DNA microarray datasets obtained under various conditions and methods. In this study, we grouped homologous genes to perform a meta-analysis of multiple vascular endothelial cell and skeletal muscle datasets. According to the t-distributed stochastic neighbor embedding (t-SNE) analysis, the changes in the gene expression pattern in vascular endothelial cells formed specific clusters. We also identified candidate genes in endothelial cells that responded to gravity. Further, we exposed human umbilical vein endothelial cells (HUVEC) to simulated microgravity (SMG) using a clinostat and measured the expression levels of the candidate genes. Gene expression analysis using qRT-PCR revealed that the expression level of the prostaglandin (PG) transporter gene <italic>SLCO2A1</italic> decreased in response to microgravity, consistent with the meta-analysis of microarray datasets. Furthermore, the direction of gravity affected the expression level of <italic>SLCO2A1</italic>, buttressing the finding that its expression was affected by gravity. These results suggest that a meta-analysis of DNA microarray datasets may help identify new target genes previously overlooked in individual microarray analyses.

DOI： 10.3389/fcell.2021.689662

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.bbrc.2021.03.077

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Language：English   Publishing type：Research paper (scientific journal)  

OBJECTIVE: Transient receptor potential vanilloid channel 2 (TRPV2) is a Ca2+ -permeable channel and plays a role in mediating intracellular Ca2+ current via mechanical stimuli. This study was undertaken to examine the expression and role of TRPV2 in adult articular cartilage and the development of osteoarthritis (OA). METHODS: We examined TRPV2 expression in mouse and human articular cartilage. We analyzed the development of OA in Col2a1-CreERt2 ;Trpv2fl/fl mice and Trpv2fl/fl littermates in the resection of the medial meniscus and medial collateral ligament model (n = 5 each), the destabilization of the medial meniscus model (n = 5 each), and the aging mouse model (n = 8-9 each). We examined marker protein expression in these joints, Ca2+ influx by mechanical stimuli, and downstream pathways in vitro. RESULTS: TRPV2 was expressed in mouse and human articular cartilage and ectopic ossification lesions. In all mouse models of OA examined, Col2a1-CreERt2 ;Trpv2fl/fl mice were observed to have enhanced degradation of articular cartilage accompanied by decreased expression of lubricin/Prg4, and marked formation of periarticular ectopic ossification. Mechanical stress-induced Ca2+ influx was decreased by Trpv2 knockout (KO). Prg4 induction by fluid-flow shear stress was diminished in Trpv2-KO mouse chondrocytes, and this was mediated by the Ca2+ /calmodulin-dependent protein kinase kinase-cyclic AMP response element binding protein axis. Hypertrophic differentiation was enhanced in Trpv2-KO mouse chondrocytes. Increased activity of calcineurin and nuclear translocation of nuclear factor in activated T cells 1 induced by fluid-flow shear stress or TRP agonist treatment was reversed by Trpv2 knockout. CONCLUSION: Our findings demonstrate regulation of articular cartilage by TRPV2 through Prg4 induction and suppression of ectopic ossification.

DOI： 10.1002/art.41684

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1186/s12576-021-00809-x

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Other Link： https://link.springer.com/article/10.1186/s12576-021-00809-x/fulltext.html

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1186/s12576-021-00809-x

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Other Link： https://link.springer.com/article/10.1186/s12576-021-00809-x/fulltext.html

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1186/s12576-021-00809-x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.mex.2021.101404

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Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Lifestyle changes, such as overeating and underexercising, can increase the risk of prediabetes. Diabetes is one of the leading causes of atherosclerosis, and recently it became clear that the pathophysiology of atherosclerosis progresses even before the onset of diabetic symptoms. In addition to changes in platelets and leukocytes in the hyperglycemic state and damage to vascular endothelial cells, extracellular vesicles and microRNAs were found to be involved in the progression of prediabetes atherosclerosis. This review discusses the cellular and molecular mechanisms of these processes, with an intention to enable a comprehensive understanding of the pathophysiology of prediabetes and atherosclerosis.

DOI： 10.3390/ijms22084108

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Reactive oxygen species (ROS) plays a role in intracellular signal transduction under physiological conditions while also playing an essential role in diseases such as hypertension, ischemic heart disease, and diabetes, as well as in the process of aging. The influence of ROS has some influence on the frequent occurrence of cardiovascular diseases (CVD) in diabetic patients. In this review, we considered the pathophysiological relationship between diabetes and CVD from the perspective of ROS. In addition, considering organ damage due to ROS elevation during ischemia–reperfusion, we discussed heart and lung injuries. Furthermore, we have focused on the transient receptor potential (TRP) channels and L-type calcium channels as molecular targets for ROS in ROS-induced tissue damages and have discussed about the pathophysiological mechanism of the injury.

DOI： 10.3389/fcvm.2021.649785

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A thrombus in a coronary artery causes ischemia, which eventually leads to myocardial infarction (MI) if not removed. However, removal generates reactive oxygen species (ROS), which causes ischemia–reperfusion (I/R) injury that damages the tissue and exacerbates the resulting MI. The mechanism of I/R injury is currently extensively understood. However, supplementation of exogenous antioxidants is ineffective against oxidative stress (OS). Enhancing the ability of endogenous antioxidants may be a more effective way to treat OS, and exosomes may play a role as targeted carriers. Exosomes are nanosized vesicles wrapped in biofilms which contain various complex RNAs and proteins. They are important intermediate carriers of intercellular communication and material exchange. In recent years, diagnosis and treatment with exosomes in cardiovascular diseases have gained considerable attention. Herein, we review the new findings of exosomes in the regulation of OS in coronary heart disease, discuss the possibility of exosomes as carriers for the targeted regulation of endogenous ROS generation, and compare the advantages of exosome therapy with those of stem-cell therapy. Finally, we explore several miRNAs found in exosomes against OS.

DOI： 10.3390/ijms22041729

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Rab11b, abundantly enriched in endocytic recycling compartments, is required for the establishment of the machinery of vesicle trafficking. Yet, no report has so far characterized the biological function of Rab11b in osteoclastogenesis. Using in vitro model of osteoclasts differentiated from murine macrophages like RAW-D cells or bone marrow-derived macrophages, we elucidated that Rab11b served as an inhibitory regulator of osteoclast differentiation sequentially via (i) abolishing surface abundance of RANK and c-Fms receptors; and (ii) attenuating nuclear factor of activated T-cells c1 (NFATc-1) upstream signaling cascades, following RANKL stimulation. Rab11b was localized in early and late endosomes, Golgi complex, and endoplasmic reticulum; moreover, its overexpression enlarged early and late endosomes. Upon inhibition of lysosomal function by a specific blocker, chloroquine (CLQ), we comprehensively clarified a novel function of lysosomes on mediating proteolytic degradation of c-Fms and RANK surface receptors, drastically ameliorated by Rab11b overexpression in RAW-D cell-derived osteoclasts. These findings highlight the key role of Rab11b as an inhibitor of osteoclastogenesis by directing the transport of c-Fms and RANK surface receptors to lysosomes for degradation via the axis of early endosomes-late endosomes-lysosomes, thereby contributing towards the systemic equilibrium of the bone resorption phase.

DOI： 10.3390/ijms21249352

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

Tendons and ligaments are pivotal connective tissues that tightly connect muscle and bone. In this study, we developed a novel approach to generate tendon/ligament-like tissues with a hierarchical structure, by introducing the tendon/ligament-specific transcription factor Mohawk (MKX) into the mesenchymal stem cell (MSC) line C3H10T1/2 cells, and by applying an improved three-dimensional (3D) cyclic mechanical stretch culture system. In our developed protocol, a combination of stableMkxexpression and cyclic mechanical stretch synergistically affects the structural tendon/ligament-like tissue generation and tendon related gene expression. In a histological analysis of these tendon/ligament-like tissues, an organized extracellular matrix (ECM), containing collagen type III and elastin, was observed. Moreover, we confirmed thatMkxexpression and cyclic mechanical stretch, induced the alignment of structural collagen fibril bundles that were deposited in a fibripositor-like manner during the generation of our tendon/ligament-like tissues. Our findings provide new insights for the tendon/ligament biomaterial fields.

DOI： 10.3389/fcell.2020.00307

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MyJove Corporation  

DOI： 10.3791/61104

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Mechanical stimulation is well known to be important for maintaining tissue and organ homeostasis. Here, we found that hydrostatic pressure induced nuclear translocation of a forkhead box O (FOXO) transcription factor DAF-16, in C. elegans within minutes, whereas the removal of this pressure resulted in immediate export of DAF-16 to the cytoplasm. We also monitored DAF-16-dependent transcriptional changes by exposure to 1 MPa pressure for 5 min, and found significant changes in collagen and other genes in a DAF-16 dependent manner. Lifespan was markedly prolonged with exposure to cyclic pressure treatment (1 MPa once a day for 5 min from L1 larvae until death). Furthermore, age-dependent decline in locomotor activity was suppressed by the treatment. In contrast, the nuclear translocation of the yes-associated protein YAP-1 was not induced under the same pressure conditions. Thus, moderate hydrostatic pressure improves ageing progression through activation of DAF-16/FOXO in C. elegans.

DOI： 10.1016/j.bbrc.2020.01.047

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Heart failure is the major cause of death for muscular dystrophy patients, however, the molecular pathomechanism remains unknown. Here, we show the detailed molecular pathogenesis of muscular dystrophy-associated cardiomyopathy in mice lacking the fukutin gene (Fktn), the causative gene for Fukuyama muscular dystrophy. Although cardiac Fktn elimination markedly reduced α-dystroglycan glycosylation and dystrophin-glycoprotein complex proteins in sarcolemma at all developmental stages, cardiac dysfunction was observed only in later adulthood, suggesting that membrane fragility is not the sole etiology of cardiac dysfunction. During young adulthood, Fktn-deficient mice were vulnerable to pathological hypertrophic stress with downregulation of Akt and the MEF2-histone deacetylase axis. Acute Fktn elimination caused severe cardiac dysfunction and accelerated mortality with myocyte contractile dysfunction and disordered Golgi-microtubule networks, which were ameliorated with colchicine treatment. These data reveal fukutin is crucial for maintaining myocyte physiology to prevent heart failure, and thus, the results may lead to strategies for therapeutic intervention.

DOI： 10.1038/s41467-019-13623-2

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The synovial fluids of patients with osteoarthritis (OA) contain elevated levels of inflammatory cytokines, which induce the expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and of the matrix metalloproteinase (MMP) in chondrocytes. Mechanical strain has varying effects on organisms depending on the strength, cycle, and duration of the stressor; however, it is unclear under inflammatory stimulation how mechanical strain act on. Here, we show that mechanical strain attenuates inflammatory cytokine-induced expression of matrix-degrading enzymes. Cyclic tensile strain (CTS), as a mechanical stressor, attenuated interleukin (IL)-1β and tumor necrosis factor (TNF)-α-induced mRNA expression of ADAMTS4, ADAMTS9, and MMP-13 in normal chondrocytes (NHAC-kn) and in a chondrocytic cell line (OUMS-27). This effect was abolished by treating cells with mechano-gated channel inhibitors, such as gadolinium, transient receptor potential (TRP) family inhibitor, ruthenium red, and with pharmacological and small interfering RNA-mediated TRPV1 inhibition. Furthermore, nuclear factor κB (NF-κB) translocation from the cytoplasm to the nucleus resulting from cytokine stimulation was also abolished by CTS. These findings suggest that mechanosensors such as the TRPV protein are potential therapeutic targets in treating OA.

DOI： 10.1016/j.yexcr.2019.111556

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DOI： 10.1016/j.bbrc.2019.09.119

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We investigated the relationship between human sperm rheotaxis and motile sperm trajectories by using poly-(dimethylsiloxane) (PDMS)-based cylindrical microfluidic channels with inner diameters of 100 μm, 50 μm, and 70 μm, which corresponded to the inner diameter of the human isthmus, the length of a sperm and a diameter intermediate between the two, respectively. We counted the number of rheotaxic sperm and sperm with spiral motion. We also analyzed motile sperm trajectories. As the cylindrical channel diameter was decreased, the percentage of sperm cells exhibiting rheotaxis, the percentage of sperm cells exhibiting spiral motion, the frequency-to-diameter ratio of the sperm cells' spiral trajectories, and the surface area of the microfluidic channel increased, while the flagellar motion at the channel wall decreased. The percentage of sperm exhibiting a spiral trajectory and the frequency-to-diameter ratio of the sperm cells' spiral trajectories were thus affected by the channel diameter. Our findings suggest that the oviduct structure affects the swimming properties of sperm cells, guiding them from the uterus to the ampulla for egg fertilization. These results could contribute to the development of motile sperm-sorting microfluidic devices for assisted reproductive technologies.

DOI： 10.18926/AMO/56863

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The application of mechanical stimuli to cells often induce increases in intracellular calcium, affecting the regulation of a variety of cell functions. Although the mechanism of mechanotransduction-induced calcium increases has not been fully resolved, the involvement of mechanosensitive ion channels in the plasma membrane and the endoplasmic reticulum has been reported. Here, we demonstrate that voltage-gated L-type calcium channels play a critical role in the mechanosensitive calcium response in H9c2 rat cardiomyocytes. The intracellular calcium level in H9c2 cells increased in a reproducible dose-dependent manner in response to uniaxial stretching. The stretch-activated calcium response (SICR) completely disappeared in calcium-free medium, whereas thapsigargin and cyclopiazonic acid, inhibitors of sarcoendoplasmic reticulum calcium ATPase, partially reduced the SICR. These findings suggest that both calcium influx across the cell membrane and calcium release from the sarcoendoplasmic reticulum are involved in the SICR. Nifedipine, diltiazem, and verapamil, inhibitors of L-type calcium channels, reduced the SICR in a dose-dependent manner. Furthermore, small interfering RNA against the L-type calcium channel α1c subunit diminished the SICR dramatically. Nifedipine also diminished the mechanosensitivity of Langendorff-perfused rat heart. These results suggest that the SICR in H9c2 cardiomyocytes involves the activation of L-type calcium channels and subsequent calcium release from the sarcoendoplasmic reticulum.

DOI： 10.1016/j.ceca.2019.02.008

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DOI： 10.1016/j.bbrep.2018.09.003

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DOI： 10.1038/s41598-018-35049-4

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DOI： 10.1063/1.5040564

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Language：Japanese   Publisher：(NPO)日本歯周病学会  

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DOI： 10.1016/j.bbrc.2018.07.116

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DOI： 10.3390/cells7060062

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Tokyo  

When a cardiac muscle is held in a stretched position, its [Ca2+] transient increases slowly over several minutes in a process known as stress-induced slow increase in intracellular Ca2+ concentration ([Ca2+]i) (SSC). Transient receptor potential canonical (TRPC) 3 forms a non-selective cation channel regulated by the angiotensin II type 1 receptor (AT1R). In this study, we investigated the role of TRPC3 in the SSC. Isolated mouse ventricular myocytes were electrically stimulated and subjected to sustained stretch. An AT1R blocker, a phospholipase C inhibitor, and a TRPC3 inhibitor suppressed the SSC. These inhibitors also abolished the observed SSC-like slow increase in [Ca2+]i induced by angiotensin II, instead of stretch. Furthermore, the SSC was not observed in TRPC3 knockout mice. Simulation and immunohistochemical studies suggest that sarcolemmal TRPC3 is responsible for the SSC. These results indicate that sarcolemmal TRPC3, regulated by AT1R, causes the SSC.

DOI： 10.1007/s12576-016-0519-3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press  

Mechanical properties of cardiomyocytes from different transmural regions are heterogeneous in the left ventricular wall. The cardiomyocyte mechanical environment affects this heterogeneity because of mechano-electric feedback mechanisms. In the present study, we investigated the effects of the mechanical load (preload and afterload) on transmural differences in contraction of subendocardial (ENDO) and subepicardial (EPI) single cells isolated from the murine left ventricle. Various preloads imposed via axial stretch and afterloads (unloaded and heavy loaded conditions) were applied to the cells using carbon fiber techniques for single myocytes. To simulate experimentally obtained results and to predict mechanisms underlying the cellular response to change in load, our mathematical models of the ENDO and EPI cells were used. Our major findings are the following. Our results show that ENDO and EPI cardiomyocytes have different mechanical responses to changes in preload to the cells. Under auxotonic contractions at low preload (unstretched cells), time to peak contraction (Tmax) and the time constant of [Ca2 +]i transient decay were significantly longer in ENDO cells than in EPI cells. An increase in preload (stretched cells) prolonged Tmax in both cell types
however, the prolongation was greater in EPI cells, resulting in a decrease in the transmural gradient in Tmax at high preload. Comparing unloaded and heavy loaded (isometric) contractions of the cells we found that transmural gradient in the time course of contraction is independent of the loading conditions. Our mathematical cell models were able to reproduce the experimental results on the distinct cellular responses to changes in the mechanical load when we accounted for an ENDO/EPI difference in the parameters of cooperativity of calcium activation of myofilaments.

DOI： 10.1016/j.yjmcc.2017.12.001

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier Ltd  

Mitochondria are an important source of reactive oxygen species (ROS). Although it has been reported that myocardial stretch increases cellular ROS production by activating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2), referred to as X-ROS signalling, the involvement of mitochondria in X-ROS is not clear. Mitochondria are organelles that generate adenosine triphosphate (ATP) for cellular energy needs, which are mechanical-load-dependent. Therefore, it would not be surprising if these organelles had mechano-sensitive functions associated with stretch-induced ROS production. In the present study, we investigated the relation between X-ROS and mitochondrial stretch-sensitive responses in isolated mouse cardiac myocytes. The cells were subjected to 10% axial stretch using computer-controlled, piezo-manipulated carbon fibres attached to both cell ends. Cellular ROS production and mitochondrial membrane potential (Δψm) were assessed optically by confocal microscopy. The axial stretch increased ROS production and hyperpolarised Δψm. Treatment with a mitochondrial metabolic uncoupler, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), at 0.5 μM did not suppress stretch-induced ROS production, whereas treatment with a respiratory Complex III inhibitor, antimycin A (5 μM), blunted the response. Although NOX inhibition by apocynin abrogated the stretch-induced ROS production, it did not suppress stretch-induced hyperpolarisation of Δψm. These results suggest that stretch causes activation of the respiratory chain to hyperpolarise Δψm, followed by NOX activation, which increases ROS production.

DOI： 10.1016/j.pbiomolbio.2017.05.015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier Ltd  

Transient receptor potential (TRP) channels constitute a large family of versatile multi-signal transducers. In particular, TRP canonical (TRPC) channels are known as receptor-operated, non-selective cation channels. TRPC3 and TRPC6, two members in the TRPC family, are highly expressed in the heart, and participate in the pathogenesis of cardiac hypertrophy and heart failure as a pathological response to chronic mechanical stress. In the pathological response, myocardial stretch increases intracellular Ca2+ levels and activates nuclear factor of activated T cells to induce cardiac hypertrophy. Recent studies have revealed that TRPC3 and TRPC6 also contribute to the physiological stretch-induced slow force response (SFR), a slow increase in the Ca2+ transient and twitch force during stretch. In the physiological response, a stretch-induced increase in intracellular Ca2+ mediated by TRPC3 and TRPC6 causes the SFR. We here overview experimental evidence of the involvement of TRPC3 and TRPC6 in cardiac physiology and pathophysiology in response to stretch.

DOI： 10.1016/j.pbiomolbio.2017.06.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier Ltd  

The electrical and mechanical functions of cardiomyocytes differ in relation to the spatial locations of cells in the ventricular wall. This physiological heterogeneity may change under pathophysiological conditions, providing substrates for arrhythmia and contractile dysfunctions. Previous studies have reported distinctions in the electrophysiological and mechanical responses to ischemia of unloaded subendocardial (ENDO) and subepicardial (EPI) single cardiomyocytes. In this paper, we briefly recapitulated the available experimental data on the ischemia effects on the transmural cellular gradient in the heart ventricles and for the first time evaluated the preload-dependent changes in passive and active forces in ENDO and EPI cardiomyocytes isolated from mouse hearts subjected to simulated ischemia. Combining the results obtained in mechanically loaded contracting cardiomyocytes with data from previous studies, we showed that left ventricular ENDO and EPI cardiomyocytes are different in their mechanical responses to metabolic inhibition. Simulated ischemia showed opposite effects on the stiffness of ENDO and EPI cells and greatly prolonged the time course of contraction in EPI cells than in ENDO cells, thereby changing the normal transmural gradient in the cellular mechanics.

DOI： 10.1016/j.pbiomolbio.2017.05.011

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Language：English   Publishing type：Research paper (international conference proceedings)  

DOI： 10.1093/eurheartj/ehx501.45

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Language：English   Publisher：MDPI AG  

Long-term habitation in space leads to physiological alterations such as bone loss, muscle atrophy, and cardiovascular deconditioning. Two predominant factors-namely space radiation and microgravity-have a crucial impact on oxidative stress in living organisms. Oxidative stress is also involved in the aging process, and plays important roles in the development of cardiovascular diseases including hypertension, left ventricular hypertrophy, and myocardial infarction. Here, we discuss the effects of space radiation, microgravity, and a combination of these two factors on oxidative stress. Future research may facilitate safer living in space by reducing the adverse effects of oxidative stress.

DOI： 10.3390/ijms18071426

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

DOI： 10.1016/j.bpj.2016.11.1471

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Language：Japanese   Publisher：(一社)日本生理学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACAD SCIENCES CZECH REPUBLIC, INST PHYSIOLOGY  

Various types of mechanosensitive ion channels, including cationic stretch-activated channels (SACNS) and stretch-activated BKca (SAKca) channels, modulate heart rhythm. Bepridil has been used as an antiarrhythmic drug with multiple pharmacological effects; however, whether it is effective for mechanically induced arrhythmia has not been well investigated. To test the effects of Bepridil on SAKca channels activity, cultured chick embryonic ventricular myocytes were used for single-channel recordings. Bepridil significantly reduced the open probability of the SAKca channel (P-O). Next, to test the effects of bepridil on stretch-induced extrasystoles (SIE), we used an isolated 2-week-old Langendorff-perfused chick heart. The left ventricle (LV) volume was rapidly changed, and the probability of SIE was calculated in the presence and absence of bepridil, and the effect of the drug was compared with that of Gadolinium (Gd3+). Bepridil decreased the probability of SIE despite its suppressive effects on SAKca channel activity. The effects of Gd3+, which blocks both SAKca and SACNS, on the probability of SIE were the same as those of bepridil. Our results suggest that bepridil blocks not only SAKca channels but possibly also blocks SACNS, and thus decreases the stretch-induced cation influx (stabilizing membrane potential) to compensate and override the effects of the decrease in outward SAKca current (destabilizing membrane potential).

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Language：English   Publishing type：Part of collection (book)   Publisher：wiley  

Long-term exposure to pressure/volume overload of the heart due to hypertension or aortic stenosis induces pathological hypertrophy and remodeling, and eventually causes heart failure. This process accompanies pathological phenomena such as inflammation, apoptosis, and arrhythmia. On the other hand, physical exercise promotes physiological hypertrophy and remodeling of the heart, thereby enhancing cardiac performance. Interestingly, recent research has suggested that enhancement of heart function is a result not only of physiological hypertrophy of pre-existing cardiomyocytes, but also of cardiac stem cell activation and myocyte formation. Furthermore, exercise improves exacerbated cardiac function in heart diseases, including dilated cardiomyopathy (DCM) and myocardial infarction (MI). The suggested mechanisms of action of exercise remedies for these diseases include angiogenesis, cardiac resynchronization, and inhibition of inflammatory cytokines. However, excessive exercise can cause arrhythmogenic remodeling and sudden death. In this chapter, we elaborate the mechanisms of hypertrophy and remodeling under physiological and pathological conditions and discuss future directions for coping with pathological hypertrophy and remodeling.

DOI： 10.1002/9781118966174.ch11

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Aims Although increased Na+/Ca2+ exchanger 1 (NCX1) expression is observed during heart failure (HF), the pathological role of NCX1 during the progression of HF remains unclear. We examined alterations of NCX1 expression and activity in hearts after transverse aortic constriction (TAC) surgery and explored whether NCX1 influences pressure overload-induced pathological cardiac remodelling.
Methods and result We generated novel transgenic mice in which NCX1 expression is controlled by a cardiac-specific, doxycycline (DOX)-dependent promoter. In the absence of DOX, TAC surgery caused substantial chamber dilation with a gradual decrease in contractility by 16 weeks. Cardiomyocytes showed a decline in contractility with abnormal Ca2+ handling during excitation-contraction (E-C) coupling. Reduced NCX1 activity was observed 8 weeks after TAC and was still apparent at 17 weeks. Induced NCX1 overexpression by DOX treatment starting 8 weeks after TAC returned NCX1 activity to pre-TAC levels and prevented chamber dilation with cardiac dysfunction. DOX treatment not only upregulated NCX1 expression in TAC-operated hearts but also returned L-type Ca2+ channel and sarcoplasmic reticulum (SR) Ca2+ ATPase expression levels to those in sham-operated hearts. In DOX-treated myocytes, contractility, T-tubule integrity, synchrony of Ca2+ release from the SR, and Ca2+ handling during E-C coupling was preserved 16 weeks after TAC surgery. In addition, DOX treatment attenuated the down-regulation of survival signalling and up-regulation of apoptosis signalling 16 weeks after TAC surgery.
ConclusionaEuro integral Induced overexpression of NCX1 attenuated pressure overload-induced pathological cardiac remodelling. Thus, maintaining NCX1 activity may be a potential therapeutic strategy for preventing the progression of HF.

DOI： 10.1093/cvr/cvw113

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Language：English   Publishing type：Research paper (scientific journal)  

AIMS/INTRODUCTION: Dental pulp stem cells (DPSCs) are thought to be an attractive candidate for cell therapy. We recently reported that the transplantation of DPSCs increased nerve conduction velocity and nerve blood flow in diabetic rats. In the present study, we investigated the immunomodulatory effects of DPSC transplantation on diabetic peripheral nerves. MATERIALS AND METHODS: DPSCs were isolated from the dental pulp of Sprague-Dawley rats and expanded in culture. Eight weeks after the streptozotocin injection, DPSCs were transplanted into the unilateral hindlimb skeletal muscles. Four weeks after DPSC transplantation, neurophysiological measurements, inflammatory gene expressions and the number of CD68-positive cells in sciatic nerves were assessed. To confirm the immunomodulatory effects of DPSCs, the effects of DPSC-conditioned media on lipopolysaccharide-stimulated murine macrophage RAW264.7 cells were investigated. RESULTS: Diabetic rats showed significant delays in sciatic nerve conduction velocities and decreased sciatic nerve blood flow, all of which were ameliorated by DPSC transplantation. The number of CD68-positive monocytes/macrophages and the gene expressions of M1 macrophage-expressed cytokines, tumor necrosis factor-α and interleukin-1β, were increased in the sciatic nerves of the diabetic rats. DPSC transplantation significantly decreased monocytes/macrophages and tumor necrosis factor-α messenger ribonucleic acid expression, and increased the gene expression of the M2 macrophage marker, CD206, in the sciatic nerves of the diabetic rats. The in vitro study showed that DPSC-conditioned media significantly increased the gene expressions of interleukin-10 and CD206 in lipopolysaccharide-stimulated RAW264.7 cells. CONCLUSIONS: These results suggest that DPSC transplantation promoted macrophages polarization towards anti-inflammatory M2 phenotypes, which might be one of the therapeutic mechanisms for diabetic polyneuropathy.

DOI： 10.1111/jdi.12452

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Language：English   Publishing type：Part of collection (book)   Publisher：Springer Japan  

This article describes how physical forces contribute to development, physiology, and pathology of vascular cells, focusing on endothelial cells and vascular smooth muscle cells. Based on these basic understandings of the mechanobiology, we discuss mechanomedicine, an application of the mechanobiology to medicine. Basic knowledge about cellular responses, such as cellular signal transduction pathway, gene expression, and cytoskeletal remodeling, to mechanical stimuli is important for understanding the pathology of vascular diseases including atherosclerosis. Introducing the knowledge of the vascular mechanobiology will not only contribute to the development of regenerative medicine using pluripotent stem cells but also provide a way to prevent diseases caused by thromboembolisms, such as myocardial and cerebral infarctions.

DOI： 10.1007/978-4-431-54801-0_15

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DOI： 10.1371/journal.pone.0141989

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

The liquid junction potential (LJP), the phenomenon that occurs when two electrolyte solutions of different composition come into contact, prevents accurate measurements in potentiometry. The effect of the LJP is usually remarkable in measurements of diluted solutions with low buffering capacities or low ion concentrations. Our group has constructed a simple method to eliminate the LJP by exerting spatiotemporal control of a liquid junction (LJ) formed between two solutions, a sample solution and a baseline solution (BLS), in a flow-through-type differential pH sensor probe. The method was contrived based on microfluidics. The sensor probe is a differential measurement system composed of two ion-sensitive field-effect transistors (ISFETs) and one Ag/AgCl electrode. With our new method, the border region of the sample solution and BLS is vibrated in order to mix solutions and suppress the overshoot after the sample solution is suctioned into the sensor probe. Compared to the conventional method without vibration, our method shortened the settling time from over two min to 15 s and reduced the measurement error by 86% to within 0.060 pH. This new method will be useful for improving the response characteristics and decreasing the measurement error of many apparatuses that use LJs.

DOI： 10.3390/s150407898

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Ischemic heart disease still remains the most common cause of cardiac death. During ischemia-reperfusion (I/R), reactive oxygen species (ROS) are produced in excess in cardiac tissue, where they induce cell death. Our previous study showed that 9-phenanthrol (9-Phe), a specific inhibitor of the TRPM4 channel, preserves cardiac contractile function and protects the heart from I/R injury-related infarction in the excised rat heart. Accordingly, we hypothesized that TRPM4 channels are involved in the 9-Phe-mediated cardioprotection against ROS-induced injury. In rats, intravenous 9-Phe mitigated the development of myocardial infarction caused by the occlusion of the left anterior descending artery. Immunohistochemical analysis indicated that TRPM4 proteins are expressed in ventricular myocytes susceptible to I/R injury. Hydrogen peroxide (H2O2) is among the main ROS overproduced during I/R. In the cardiomyocyte cell line H9c2, pretreatment with 9-Phe prevented cell death induced by conditions mimicking I/R, namely 200 mu M H2O2 and hypoxia-reoxygenation. Gene silencing of TRPM4 preserved the viability of H9c2 cardiomyocytes exposed to 200 mu M H2O2. These results suggest that the cardioprotective effects of 9-Phe are mediated through the inhibition of the TRPM4 channels.

DOI： 10.1371/journal.pone.0121703

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER THORACIC SOC  

Airway smooth muscle (ASM) cells within the airway walls are continually exposed to mechanical stimuli, and exhibit various functions in response to these mechanical stresses. ATP acts as an extracellular mediator in the airway. Moreover, extracellular ATP is considered to play an important role in the pathophysiology of asthma and chronic obstructive pulmonary disease. However, it is not known whether ASM cells are cellular sources of ATP secretion in the airway. We therefore investigated whether mechanical stretch induces ATP release from ASM cells. Mechanical stretch was applied to primary human ASM cells cultured on a silicone chamber coated with type I collagen using a stretching apparatus. Concentrations of ATP in cell culture supernatants measured by luciferin-luciferase bioluminescence were significantly elevated by cyclic stretch (12 and 20% strain). We further visualized the stretch-induced ATP release from the cells in real time using a luminescence imaging system, while acquiring differential interference contrast cell images with infrared optics. Immediately after a single uniaxial stretch for 1 second, strong ATP signals were produced by a certain population of cells and spread to surrounding spaces. The cyclic stretch-induced ATP release was significantly reduced by inhibitors of Ca2+-dependent vesicular exocytosis, 1,2-bis(o-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid tetraacetoxymethyl ester, monensin, N-ethylmaleimide, and bafilomycin. In contrast, the stretch-induced ATP release was not inhibited by a hemichannel blocker, carbenoxolone, or blockade of transient receptor potential vanilloid 4 by short interfering RNA transfection or ruthenium red. These findings reveal a novel property of ASM cells: mechanically induced ATP release may be a cellular source of ATP in the airway.

DOI： 10.1165/rcmb.2014-0008OC

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca2+ concentration ([Ca2+](i)) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10-30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca2+](i) transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca2+](i) The stretch-induced [Ca2+](i) elevation was attenuated in Ca2+-free solution. In contrast, the increase of [Ca2+](i) by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd3+, ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca2+](i) elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca2+ influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP. (C) 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2014.09.063

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Soc. of Med. Electronics and Biol. Engineering  

Na+/Ca&lt
inf&gt
2+&lt
/inf&gt
exchanger (NCX1) is essential Ca&lt
inf&gt
2+&lt
/inf&gt
regulator of myocyte Ca&lt
inf&gt
2+&lt
/inf&gt
homeostasis and specially localized at transverse tubules (T-tubules) membrane. T-tubules are invaginations of the sarcolemma and critical for myocyte contraction, especially as the main site of excitation-contraction coupling. Therefore, T-tubule disorganization is linked to decreased contractility in heart failure, but the molecular mechanism is not clear. We analyzed the alteration of T-tubule structure and Ca&lt
inf&gt
2+&lt
/inf&gt
handling during the progression of heart failure after transverse aortic constriction (TAC)-surgery, using cardiac-specific and inducible NCX1 transgenic mice. In progression of cardiac dysfunction, sarcoplasmic reticulum Ca&lt
inf&gt
2+&lt
/inf&gt
ATPase and NCX1 activity were down-regulated before T-tubule disorganization. The inducing NCX1 overexpression after TAC-surgery prevented T-tubule disorganization and contractile dysfunction under prolonged pressure-overload, with improvement of myocyte Ca&lt
inf&gt
2+&lt
/inf&gt
handling. These results suggest that local Ca&lt
inf&gt
2+&lt
/inf&gt
control beneath the T-tubule membrane is crucial for the maintenance of myocyte structure and function, in which NCX1 has a pivotal role.

DOI： 10.11239/jsmbe.52.O-212

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Soc. of Med. Electronics and Biol. Engineering  

Myocardium contracts against ventricular wall stretch that comes along with ventricular filling. Mitochondria generate required ATP for myocardial contraction. It is well known that mitochondrial ATP production process is one of the sources of reactive oxygen species (ROS). ROS are known as toxic molecules, but also important physiological regulators of intracellular signaling pathways. In the present study, we investigate the relation between myocardial stretch and mitochondrial ROS production, and discuss the role of mitochondria on myocardial response to stretch. Isolated mouse ventricular myocytes were exposed to 10% axial stretch using carbon fiber technique. ROS production was studied using DCF-loaded cells. Axial stretch significantly increased ROS production. Applying 5 μM mitochondrial metabolic uncoupler FCCP blunted the response, indicating mitochondrial ROS production is stretch-sensitive. The present results suggest that stretch enhances electron transport chain to prepare for the more ATP production for the more preloaded, namely, the more energy-consuming contraction.

DOI： 10.11239/jsmbe.52.SY-44

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Soc. of Med. Electronics and Biol. Engineering  

Low-cost paper-based diagnostic device for human sperm motility has developed to know sperm motility without consulting fertility clinics. We compared the signal pattern of the paper-based sperm motility assay using 4-channel pattern (cross) and 2-channel pattern (line) papers, and investigated difference in signal patterns using some tetrazorium salts for the assay. Using these two paper patterns, we can examine whether human sperm motility in semen is more than 50% or 0%. XTT and WST-8 reagents treated paper-based device can be used. We concluded that MTT treated line pattern paper is suitable for the purpose to reduce the cost of the device.

DOI： 10.11239/jsmbe.52.O-551

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The previously reported pressure-volume (PV) relationship in frog hearts shows that end-systolic PV relation (ESPVR) is load dependent, whereas ESPVR in canine hearts is load independent. To study intrinsic cardiac mechanics in detail, it is desirable to study mechanics in a single isolated cardiomyocyte that is free from interstitial connective tissue. Previous single cell mechanics studies used a pair of carbon fibers (CF) attached to the upper surface of opposite cell ends to stretch cells. These studies showed that end-systolic force-length (FL) relation (ESFLR) is load independent. However, the range of applicable mechanical load using the conventional technique is limited because of weak cell-CF attachment. Therefore, the behavior of ESFLR in single cells under physiologically possible conditions of greater load is not yet well known. To cover wider loading range, we contrived a new method to hold cell-ends more firmly using two pairs of CF attached to both upper and bottom surfaces of cells. The new method allowed stretching cells to 2.2 mu m or more in end-diastolic sarcomere length. ESFLR virtually behaves in a load independent manner only with end-diastolic sarcomere length less than 1.95 mu m. It exhibited clear load dependency with higher preload, especially with low afterload conditions. Instantaneous cellular elastance curves showed that decreasing afterload enhanced relaxation and slowed time to peak elastance, as previously reported. A simulation study of a mathematical model with detailed description of thin filament activation suggested that velocity dependent thin filament inactivation is crucial for the observed load dependent behaviors and previously reported afterload dependent change in Ca2+ transient shape. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.pbiomolbio.2014.06.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Conventional self-assembling peptide hydrogels are effective as topical hemostatic agents. However, there is a possibility to harm living tissues due to their low pH. The aim of the present study was to demonstrate the efficacy of SPG-178, a neutral self-assembling peptide hydrogel, as a topical hemostatic agent. First, we measured the bleeding duration of incisions made on rat livers after application of SPG-178 (1.0% w/v), SPG-178 (1.5% w/v), RADA16 (1.0% w/v), and saline (n = 12/group). Second, we observed the bleeding surfaces by transmission electron microscopy immediately after hemostasis. Third, we measured the elastic and viscous responses (G' and G '', respectively) of the hydrogels using a rheometer. Our results showed that bleeding duration was significantly shorter in the SPG-178 group than in the RADA16 group and that there were no significant differences in transmission electron microscopy findings between the groups. The greater the G' value of a hydrogel, the shorter was the bleeding duration. We concluded that SPG-178 is more effective and has several advantages: it is non-biological, transparent, nonadherent, and neutral and can be sterilized by autoclaving.

DOI： 10.1371/journal.pone.0102778

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

The genetic basis of hypoplastic left heart syndrome (HLHS) remains unknown, and the lack of animal models to reconstitute the cardiac maldevelopment has hampered the study of this disease. This study investigated the altered control of transcriptional and epigenetic programs that may affect the development of HLHS by using disease-specific induced pluripotent stem (iPS) cells. Cardiac progenitor cells (CPCs) were isolated from patients with congenital heart diseases to generate patient-specific iPS cells. Comparative gene expression analysis of HLHS-and biventricle (BV) heart-derived iPS cells was performed to dissect the complex genetic circuits that may promote the disease phenotype. Both HLHS-and BV heart-derived CPCs were reprogrammed to generate disease-specific iPS cells, which showed characteristic human embryonic stem cell signatures, expressed pluripotency markers, and could give rise to cardiomyocytes. However, HLHS-iPS cells exhibited lower cardiomyogenic differentiation potential than BV-iPS cells. Quantitative gene expression analysis demonstrated that HLHS-derived iPS cells showed transcriptional repression of NKX2-5, reduced levels of TBX2 and NOTCH/HEY signaling, and inhibited HAND1/2 transcripts compared with control cells. Although both HLHS-derived CPCs and iPS cells showed reduced SRE and TNNT2 transcriptional activation compared with BV-derived cells, co-transfection of NKX2-5, HAND1, and NOTCH1 into HLHS-derived cells resulted in synergistic restoration of these promoters activation. Notably, gain- and loss-of-function studies revealed that NKX2-5 had a predominant impact on NPPA transcriptional activation. Moreover, differentiated HLHS-derived iPS cells showed reduced H3K4 dimethylation as well as histone H3 acetylation but increased H3K27 trimethylation to inhibit transcriptional activation on the NKX2-5 promoter. These findings suggest that patient-specific iPS cells may provide molecular insights into complex transcriptional and epigenetic mechanisms, at least in part, through combinatorial expression of NKX2-5, HAND1, and NOTCH1 that coordinately contribute to cardiac malformations in HLHS.

DOI： 10.1371/journal.pone.0102796

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

The heart has a dynamic compensatory mechanism for haemodynamic stress. However, the molecular details of how mechanical forces are transduced in the heart are unclear. Here we show that the transient receptor potential, vanilloid family type 2 (TRPV2) cation channel is critical for the maintenance of cardiac structure and function. Within 4 days of eliminating TRPV2 from hearts of the adult mice, cardiac function declines severely, with disorganization of the intercalated discs that support mechanical coupling with neighbouring myocytes and myocardial conduction defects. After 9 days, cell shortening and Ca2+ handling by single myocytes are impaired in TRPV2-deficient hearts. TRPV2-deficient neonatal cardiomyocytes form no intercalated discs and show no extracellular Ca2+-dependent intracellular Ca2+ increase and insulin-like growth factor (IGF-1) secretion in response to stretch stimulation. We further demonstrate that IGF-1 receptor/PI3K/Akt pathway signalling is significantly downregulated in TRPV2-deficient hearts, and that IGF-1 administration partially prevents chamber dilation and impairment in cardiac pump function in these hearts. Our results improve our understanding of the molecular processes underlying the maintenance of cardiac structure and function.

DOI： 10.1038/ncomms4932

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER HEIDELBERG  

Male infertility, which amounts to half of all infertility cases, is a serious problem worldwide. The percentage of fertility-related patients in sub-Saharan African countries is higher than that for the developed countries. In low-resource countries, examination of sperm characteristics for male infertility cannot be undertaken because of poor clinical access. To evaluate male fertility in assisted reproductive medicine laboratories, the numbers of motile human sperm, the degree of sperm motility, and sperm morphology have been commonly analyzed using a microscope. It is challenging to monitor the health status of human sperm in resource-limited or remote settings for two primary reasons: (1) high capital cost (equipment for currently accepted procedural standard), and (2) complexity of the currently accepted procedural standard used to simultaneously measure human sperm concentration and motility by skillful embryologists. Determining the health status of human sperm in order to evaluate fertilization capacity using various types of low-cost, easy-to-use, and rapid devices (or systems) is a longstanding but interesting biotechnologically relevant issue in various scientific communities such as male reproduction. Furthering such efforts will inherently influence birth rate in both developed and developing nations. We have demonstrated an inexpensive but robust and easy-to-handle device for monitoring the health status of human sperm made by patterning a piece of paper and measuring the activity of a specific enzyme-a simple and elegant solution. After applying semen to the hydrophilic center circle of our patterned paper, a thiazine assay can be used to suggest sperm concentration in semen, and a tetrazolium-based colorimetric assay (MTT assay) data can be used to help estimate the percentage of motile human sperm (sperm motility) in semen based on the character that motile human sperm moved in and on the paper. Using this paper-based device, we can evaluate fertility levels without consulting doctors and use our assay to compare results with World Health Organization (WHO) reference values for sperm concentration (&gt; 2x10(7)) and motility (&gt; 50 %). The duration and cost of one entire test are 30 min and 0.1 USD, respectively. We believe that this paper-based assay system would be useful for fertility checks based on WHO references, without need of a microscope, at home. Using this assay method, males in developed or developing countries who are reluctant or unable to consult assisted reproductive technologies clinics can self-analyze their sperm characteristics. We further note that our approach adheres to WHO regulations, especially in regard to in vitro diagnostic device performance with an associated diagnostic algorithm to enhance diagnostic accuracy (compared with just one diagnostic output), and we wish to emphasize that our research could significantly advance a broad range of diagnostic developments including paper-based diagnostic devices, in vitro diagnostic devices, and diagnosis of other diseases in various divisions of translational medicine. These results, we believe, will be of interest to a wide scientific audience working in materials science (biomaterials), chemistry (analytical and clinical), lab-on-a-chip technologies (the development of diagnostic tools), reproductive medicine, bioengineering, and translational medicine.

DOI： 10.1007/s10404-014-1378-y

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Language：Japanese   Publisher：日本骨形態計測学会  

細胞内カルシウム(Ca2+)は多様な細胞機能を仲介する重要な情報伝達物質であり、Ca2+オシレーションと呼ばれる周期的なCa2+濃度の上昇様式は、骨代謝調節を含む広範な生理機構に関与すると考えられている。しかし、3次元的な骨芽細胞-骨細胞ネットワークから成り立つ骨組織中でのCa2+オシレーションの動態は、周囲の骨基質が障壁となるためこれまで不明であった。近年我々は、骨組織をまるごと生きた状態のままCa2+イメージングする事に成功し、骨芽細胞と骨細胞の自律性細胞内Ca2+オシレーションの存在と、それらの現象に対するメカニカルストレスの影響およびギャップ結合の関与について報告した。本論文では、蛍光ライブイメージングを用いた骨組織中のCa2+オシレーションについて最新の研究成果を交えて解説する。(著者抄録)

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Intracellular calcium (Ca2+) is an important secondary messenger that modulates many cellular processes. Its oscillatory signaling is considered to participate in the regulation of many different cell functions, including bone metabolism. However, it is not entirely clear whether Ca2+ oscillations occur between osteoblasts and osteocytes in integrated bone tissues because of the complex mineralized matrices surrounding bone cells. To address this issue, we have recently developed a novel ex vivo realtime imaging system, which made it possible to observe repetitive and autonomous oscillations in the intracellular Ca2+ concentration ([Ca2+]i) in intact chick calvarial explants. This system revealed that Ca2+ release from intracellular stores plays a key role in Ca2+ oscillations in bone cells. Additionally, gap junctions are important for the maintenance of these oscillations in osteocytes but not in osteoblasts. In this review, we describe the dynamic oscillatory elevations of Ca2+ levels that occur in osteoblasts and osteocytes in living bone. © 2014 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.job.2014.03.002

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Other Link： http://orcid.org/0000-0002-4419-9643

Language：English   Publishing type：Research paper (scientific journal)  

COA-Cl (2Cl-C.OXT-A) is a recently developed adenosine-like nucleic acid analog that promotes angiogenesis via the mitogen-activated protein (MAP) kinases ERK1/2. Endothelial S1P1 receptor plays indispensable roles in developmental angiogenesis. In this study, we examined the functions of S1P1 in COA-Cl-induced angiogenic responses. Antagonists for S1P1, W146, and VPC23019, substantially but still partly inhibited the effects of COA-Cl with regard to ERK1/2 activation and tube formation in cultured human umbilical vein endothelial cells (HUVEC). Antagonists for adenosine A1 receptor and purinergic P2Y1 receptor were without effect. Genetic knockdown of S1P1 with siRNA, but not that of S1P3, attenuated COA-Cl-elicited ERK1/2 responses. The signaling properties of COA-Cl showed significant similarities to those of sphingosine 1-phosphate, an endogenous S1P1 ligand, in that both induced responses sensitive to pertussis toxin (Gα i/o inhibitor), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), (calcium chelator), and PP2 (c-Src tyrosine kinase inhibitor). COA-Cl elevated intracellular Ca(2+) concentration and induced tyrosine phosphorylation of p130Cas, a substrate of c-Src, in HUVEC. COA-Cl displaced [(3)H]S1P in a radioligand-binding competition assay in chem-1 cells overexpressing S1P1. However, COA-Cl activated ERK1/2 in CHO-K1 cells that lack functional S1P1 receptor, suggesting the presence of additional yet-to-be-defined COA-Cl target in these cells. The results thus suggest the major contribution of S1P1 in the angiogenic effects of COA-Cl. However, other mechanism such as that seen in CHO-K1 cells may also be partly involved. Collectively, these findings may lead to refinement of the design of this nucleic acid analog and ultimately to development of small molecule-based therapeutic angiogenesis.

DOI： 10.1002/prp2.68

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Language：English   Publisher：American Institute of Mathematical Sciences  

Coarse-grained molecular dynamics (CGMD) simulations are increasingly being used to analyze the behaviors of biological systems. When appropriately used, CGMD can simulate the behaviors of molecular systems several hundred times faster than elaborate all-atom molecular dynamics simulations with similar accuracy. CGMD parameters for lipids, proteins, nucleic acids, and some artificial substances such as carbon nanotubes have been suggested. Here we briefly discuss a method for CGMD system configuration and the types of analysis and perturbations that can be performed with CGMD simulations. We also describe specific examples to show how CGMD simulations have been applied to various situations, and then describe experimental results that were used to validate the simulation results. CGMD simulations are applicable to resolving problems for various biological systems.

DOI： 10.3934/biophy.2014.1.1

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/BF03405834

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Language：Japanese   Publisher：Japanese Society for Medical and Biological Engineering  

We have demonstrated an inexpensive but robust and easy-to-handle device for monitoring the health status of human sperm made by patterning a piece of paper and measuring the activity of a specific enzyme. After applying semen to the hydrophilic center circle of our patterned paper, the thizain assay could suggest sperm concentration in semen, and MTT assay data could contribute to estimating the percentage of motile human sperm in semen. Using this paper-based device, we can evaluate fertility levels without consulting doctors, and use our assay to compare results with WHO reference values for sperm concentration (10-20M) and motility (more than 50%).We believe that this paper-based assay system would be useful for fertility checks based on WHO references, without need of a microscope, and at home. Using this assay method, males in developed or developing countries who are reluctant or unable to consult ART clinics can self-analyze their sperm characteristics.

DOI： 10.11239/jsmbe.52.O-551

CiNii Article

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Language：Japanese   Publisher：Japanese Society for Medical and Biological Engineering  

Na⁺/Ca²⁺ exchanger (NCX1) is essential Ca²⁺ regulator of myocyte Ca²⁺ homeostasis and specially localized at transverse tubules (T-tubules) membrane. T-tubules are invaginations of the sarcolemma and critical for myocyte contraction, especially as the main site of excitation-contraction coupling. Therefore, T-tubule disorganization is linked to decreased contractility in heart failure, but the molecular mechanism is not clear. We analyzed the alteration of T-tubule structure and Ca2+ handling during the progression of heart failure after transverse aortic constriction (TAC)-surgery, using cardiac-specific and inducible NCX1 transgenic mice. In progression of cardiac dysfunction, sarcoplasmic reticulum Ca²⁺ ATPase and NCX1 activity were down-regulated before T-tubule disorganization. The inducing NCX1 overexpression after TAC-surgery prevented T-tubule disorganization and contractile dysfunction under prolonged pressure-overload, with improvement of myocyte Ca²⁺ handling. These results suggest that local Ca²⁺ control beneath the T-tubule membrane is crucial for the maintenance of myocyte structure and function, in which NCX1 has a pivotal role.

DOI： 10.11239/jsmbe.52.O-212

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Azelnidipine and amlodipine are dihydropyridine-type Ca2+ channel blockers for the treatment of hypertension. Although these drugs have high vasoselectivity and small negative inotropic effects in vivo, little is known regarding their direct effects on cellular contractility without humoral regulation or the additive effects of these drugs with other antihypertensive drugs on myocardial contractility. To investigate the effects of Ca2+ channel blockers on single cell mechanics, mouse cardiomyocytes were enzymatically isolated, and a pair of carbon fibers was attached to opposite cell-ends to stretch the cells. Cells were paced at 4 Hz superfused in normal Tyrode solution at 37 C. Cell length and active/passive force calculated from carbon fiber bending were recorded in 6 different preload conditions. Slopes of end-systolic force length relation curves (maximum elastance) were measured as an index of contractility before and after drugs were administered. Azelniclipine at 10 nM and 100 nM did not change maximum elastance, while amlodipine at 100 nM did decrease maximum elastance. The combination of RNH-6270 (active form of angiotensin II receptor blocker, olmesartan, 10 nM) and either amlodipine (10 nM) or azelnidipine (10 nM) did not affect maximum elastance. Although both amlodipine and azelnidipine can be used safely at therapeutically relevant concentrations even in combination with olmesartan, the present results suggest that azelnidipine has a less negative inotropic action compared to amlodipine. (C) 2013 Elsevier B.V. All rights reserved,

DOI： 10.1016/j.ejphar.2013.05.030

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

A group of muscular dystrophies, dystroglycanopathy is caused by abnormalities in post-translational modifications of dystroglycan (DG). To understand better the pathophysiological roles of DG modification and to establish effective clinical treatment for dystroglycanopathy, we here generated two distinct conditional knock-out (cKO) mice for fukutin, the first dystroglycanopathy gene identified for Fukuyama congenital muscular dystrophy. The first dystroglycanopathy modelumyofiber-selective fukutin-cKO [muscle creatine kinase (MCK)-fukutin-cKO] miceushowed mild muscular dystrophy. Forced exercise experiments in presymptomatic MCK-fukutin-cKO mice revealed that myofiber membrane fragility triggered disease manifestation. The second dystroglycanopathy modelumuscle precursor cell (MPC)-selective cKO (Myf5-fukutin-cKO) miceuexhibited more severe phenotypes of muscular dystrophy. Using an isolated MPC culture system, we demonstrated, for the first time, that defects in the fukutin-dependent modification of DG lead to impairment of MPC proliferation, differentiation and muscle regeneration. These results suggest that impaired MPC viability contributes to the pathology of dystroglycanopathy. Since our data suggested that frequent cycles of myofiber degeneration/regeneration accelerate substantial and/or functional loss of MPC, we expected that protection from disease-triggering myofiber degeneration provides therapeutic effects even in mouse models with MPC defects; therefore, we restored fukutin expression in myofibers. Adeno-associated virus (AAV)-mediated rescue of fukutin expression that was limited in myofibers successfully ameliorated the severe pathology even after disease progression. In addition, compared with other gene therapy studies, considerably low AAV titers were associated with therapeutic effects. Together, our findings indicated that fukutin-deficient dystroglycanopathy is a regeneration-defective disorder, and gene therapy is a feasible treatment for the wide range of dystroglycanopathy even after disease progression.

DOI： 10.1093/hmg/ddt157

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Despite efforts to elucidate its pathophysiology, ischemia-reperfusion injury lacks an effective preventative intervention. Because transient receptor potential cation channel subfamily M member 4 (TRPM4) is functionally expressed by many cell types in the cardiovascular system and is involved in the pathogenesis of various cardiovascular diseases, we decided to assess its suitability as a target of therapy. Thus, the aim of this study was to examine the possible cardioprotective effect of 9-phenanthrol, a specific inhibitor of TRPM4. Isolated Langendorff-perfused rat hearts were pretreated with Krebs-Henseleit (K-H) solution (control), 9-phenanthrol, or 5-hydroxydecanoate (5-HD, a blocker of the ATP-sensitive potassium channel) and then subjected to global ischemia followed by reperfusion with the K-H solution. To evaluate the extent of heart damage, lactate dehydrogenase (LDH) activity in the effluent solution was measured, and the size of infarcted area of the heart was measured by 2,3,5-triphenyltetrazolium chloride staining. In controls, cardiac contractility decreased, and LDH activity and the infarcted area size increased. In contrast, in hearts pretreated with 9-phenanthrol, contractile function recovered dramatically, and the infarcted area size significantly decreased. The cardioprotective effects of 9-phenanthrol was not completely blocked by 5-HD. These findings show that 9-phenanthrol exerts a cardioprotective effect against ischemia in the isolated rat heart and suggest that its mechanism of action is largely independent of ATP-sensitive potassium channels.

DOI： 10.1371/journal.pone.0070587

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Language：Japanese   Publisher：日本骨形態計測学会  

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The focal adhesion (FA) is an integrin-based structure built in/on the plasma membrane (PM), linking the extracellular matrix to the actin stress-fibers, working as cell migration scaffolds. Previously, we proposed the archipelago architecture of the FA, in which FA largely consists of fluid membrane, dotted with small islands accumulating FA proteins: membrane molecules enter the inter-island channels in the FA zone rather freely, and the integrins in the FA-protein islands rapidly exchanges with those in the bulk membrane. Here, we examined how Rac1, a small G-protein regulating FA formation, and its activators αPIX and βPIX, are recruited to the FA zones. PIX molecules are recruited from the cytoplasm to the FA zones directly. In contrast, majorities of Rac1 molecules first arrive from the cytoplasm on the general inner PM surface, and then enter the FA zones via lateral diffusion on the PM, which is possible due to rapid Rac1 diffusion even within the FA zones, slowed only by a factor of two to four compared with that outside. The constitutively-active Rac1 mutant exhibited temporary and all-time immobilizations in the FA zone, suggesting that upon PIX-induced Rac1 activation at the FA-protein islands, Rac1 tends to be immobilized at the FA-protein islands. © 2013 Wiley Periodicals, Inc.

DOI： 10.1002/cm.21097

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Bone cells respond to mechanical stimuli by producing a variety of biological signals, and one of the earliest events is intracellular calcium ([Ca2+](i)) mobilization. Our recently developed ex vivo live [Ca2+](i) imaging system revealed that bone cells in intact bone explants showed autonomous [Ca2+](i) oscillations, and osteocytes specifically modulated these oscillations through gap junctions. However, the behavior and connectivity of the [Ca2+](i) signaling networks in mechanotransduction have not been investigated in intact bone. We herein introduce a novel fluid-flow platform for probing cellular signaling networks in live intact bone, which allows the application of capillary-driven flow just on the bone explant surface while performing real-time fluorogenic monitoring of the [Ca2+](i) changes. In response to the flow, the percentage of responsive cells was increased in both osteoblasts and osteocytes, together with upregulation of c-fos expression in the explants. However, enhancement of the peak relative fluorescence intensity was not evident. Treatment with 18 alpha-GA, a reversible inhibitor of gap junction, significantly blocked the [Ca2+](i), responsiveness in osteocytes without exerting any major effect in osteoblasts. On the contrary, such treatment significantly decreased the flow-activated oscillatory response frequency in both osteoblasts and osteocytes. The stretch-activated membrane channel, when blocked by Gd3+, is less affected in the flow-induced [Ca2+](i) response. These findings indicated that flow-induced mechanical stimuli accompanied the activation of the autonomous [Ca2+](i) oscillations in both osteoblasts and osteocytes via gap junction-mediated cell-cell communication and hemichannel. Although how the bone sense the mechanical stimuli in vivo still needs to be elucidated, the present study suggests that cell-cell signaling via augmented gap junction and hemichannel-mediated [Ca2+](i) mobilization could be involved as an early signaling event in mechanotransduction. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2012.12.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

Human embryos normally experience mechanical stimuli during development in vivo. To apply appropriate stimuli to embryos, this study group developed a tilting embryo culture system (TECS) and investigated whether it could improve the grade of fresh human embryos compared with a control static culture system. A total of 450 retrieved oocytes from 32 IVF or intracytoplasmic sperm injection cycles of 32 women were cultured for 5-6 days. Oocytes were divided randomly into TECS and control groups and then were inseminated in vitro. All embryos were evaluated at days 3 and 5 using standard grading criteria for embryo quality. The rates of fertilization per mature oocyte and high-grade cleavage-stage embryo formation in the TECS group were similar to those in the control group. The rates of blastocyst formation and of blastocysts graded 3BB or higher at day 5 were significantly higher in the TECS group than those in the control group: 45.3% (67/148) versus 32.1% (51/159) (P = 0.018) and 29.1% (43/148) versus 17.6% (28/159) (P = 0.018), respectively. The TECS group produced more high-grade blastocysts than the control group. Embryo movement or mechanical stimulation during embryo culture may be beneficial for human embryonic development. (C) 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.rbmo.2012.11.014

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT INC  

Dental pulp stem cells (DPSCs), which can differentiate into several types of cells, are subjected to mechanical stress by jaw movement and occlusal forces. In this study, we evaluated how the uniaxial mechanical stretch influences proliferation and differentiation of DPSCs. DPSCs were isolated and cultured from male Sprague-Dawley rats. Cultured DPSCs were identified by surface markers and the differentiation capabilities as adipocytes or osteoblasts. To examine the response to mechanical stress, uniaxial stretch was exposed to cultured DPSCs. We evaluated the impact of stretch on the intracellular signaling, proliferation, osteogenic differentiation, and gene expressions of DPSCs. Stretch increased the phosphorylation of Akt, ERK1/2, and p38 MAP kinase as well as the proliferation of DPSCs. The stretch-induced proliferation of DPSCs was abolished by the inhibition of the ERK pathway. On the other hand, stretch significantly decreased the osteogenic differentiation of DPSCs, but did not affect the adipogenic differentiation. We also confirmed mRNA expressions of osteocalcin and osteopontin were significantly suppressed by stretch. In conclusion, uniaxial stretch increased the proliferation of DPSCs, while suppressing osteogenic differentiation. These results suggest a crucial role of mechanical stretch in the preservation of DPSCs in dentin. Furthermore, mechanical stretch may be a useful tool for increasing the quantity of DPSCs in vitro for regenerative medicine.

DOI： 10.1089/ten.tea.2012.0099

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Biofilm formation in microfluidic channels is difficult to detect because sampling volumes are too small for conventional turbidity measurements. To detect biofilm formation, we used an ion-sensitive field-effect transistor (ISFET) measurement system to measure pH changes in small volumes of bacterial suspension. Cells of Micrococcus luteus (M. luteus) were cultured in polystyrene (PS) microtubes and polymethylmethacrylate (PMMA)-based microfluidic channels laminated with polyvinylidene chloride. In microtubes, concentrations of bacteria and pH in the suspension were analyzed by measuring turbidity and using an ISFET sensor, respectively. In microfluidic channels containing 20 mu L of bacterial suspension, we measured pH changes using the ISFET sensor and monitored biofilm formation using a microscope. We detected acidification and alkalinization phases of M. luteus from the ISFET sensor signals in both microtubes and microfluidic channels. In the alkalinization phase, after 2 day culture, dense biofilm formation was observed at the bottom of the microfluidic channels. In this study, we used an ISFET sensor to detect biofilm formation in clinical and industrial microfluidic environments by detecting alkalinization of the culture medium.

DOI： 10.3390/s130202484

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Mechanosensitivity is essential for heart function just as for all other cells and organs in the body, and it is involved in both normal physiology and diseases processes of the cardiovascular system. In this review, we have outlined the relationship between mechanosensitivity and heart physiology, including the FrankStarling law of the heart and mechanoelectric feedback. We then focused on molecules involved in mechanotransduction, particularly mechanosensitive ion channels. We have also discussed the involvement of mechanosensitivity in heart diseases, such as arrhythmias, hypertrophy and ischaemic heart disease. Finally, mechanobiology in cardiogenesis is described with regard to regenerative medicine.

DOI： 10.1111/jcmm.12027

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Language：English   Publisher：Japanese Society for Medical and Biological Engineering  

DOI： 10.11239/jsmbe.51.M-32

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The heart is an organ that is exposed to extreme dynamic mechanical stimuli. From birth till death, the heart indefinitely repeats periodic contraction and dilation, i.e., shortening and elongation of cardiomyocytes. Mechanical stretch elicits a change in heart rate and may cause arrhythmia if it is excessive. Thus, mechanosensitivity is crucial to heart function. The molecule that is substantially involved in mechanosensitivity is a stretch-activated ion channel. Among several ion channels believed to be activated by stretch in the heart, the stretch-activated K-Ca (SAKCA) channel, a member of the group of large conductance (Big Potassium, BK) channels, shows a mechanosensitive (MS) response to membrane stretch. As BK channels respond to voltage and intracellular calcium concentration with large conductance, they are considered to be involved in repolarization after depolarization. Some BK channels are known to be activated by stretch and are expressed in a number of cells, including human osteoblasts and guinea pig intestinal neurons. The SAKCA channel was found to be sensitive to stretch in the chick heart. Given that the cardiomyocyte is unremittingly exposed to contraction and dilation and that it generates action potential and its contractility is modulated by intracellular calcium concentration, the SAKCA channel, which is dependent voltage and calcium, may be involved in action potential generation. It was recently reported that a BK channel is involved in the modulation of heart rate in the mouse. Further studies regarding the role of MS BK channels, including SAKCA, in the modulation of heart rate and contractility are expected. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.pbiomolbio.2012.08.001

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DOI： 10.1016/j.cardfail.2012.08.116

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

The focal adhesion (FA) is an integrin-based structure built in/on the plasma membrane, mechanically linking the extracellular matrix with the termini of actin stress fibers, providing key scaffolds for the cells to migrate in tissues. The FA was considered as a micron-scale, massive assembly of various proteins, although its formation and decomposition occur quickly in several to several 10 s of minutes. The mechanism of rapid FA regulation has been a major mystery in cell biology. Here, using fast single fluorescent-molecule imaging, we found that transferrin receptor and Thy1, non-FA membrane proteins, readily enter the FA zone, diffuse rapidly there, and exit into the bulk plasma membrane. Integrin beta 3 also readily enters the FA zone, and repeatedly undergoes temporary immobilization and diffusion in the FA zone, whereas approximately one-third of integrin beta 3 is immobilized there. These results are consistent with the archipelago architecture of the FA, which consists of many integrin islands: the membrane molecules enter the inter-island channels rather freely, and the integrins in the integrin islands can be rapidly exchanged with those in the bulk membrane. Such an archipelago architecture would allow rapid FA formation and disintegration, and might be applicable to other large protein domains in the plasma membrane. (C) 2012 Wiley Periodicals, Inc

DOI： 10.1002/cm.21032

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Language：Japanese   Publisher：日本骨形態計測学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Bone cells form a complex three-dimensional network consisting of osteoblasts and osteocytes embedded in a mineralized extracellular matrix. Ca2+ acts as a ubiquitous secondary messenger in various physiological cellular processes and transduces numerous signals to the cell interior and between cells. However, the intracellular Ca2+ dynamics of bone cells have not been evaluated in living bone. In the present study, we developed a novel ex-vivo live Ca2+ imaging system that allows the dynamic intracellular Ca2+ concentration ([Ca2+](i)) responses of intact chick calvaria explants to be observed without damaging the bone network. Our live imaging analysis revealed for the first time that both osteoblasts and osteocytes display repetitive and autonomic [Ca2+](i) oscillations ex vivo. Thapsigargin, an inhibitor of the endoplasmic reticulum that induces the emptying of intracellular Ca2+ stores, abolished these [Ca2+](i) responses in both osteoblasts and osteocytes, indicating that Ca2+ release from intracellular stores plays a key role in the [Ca2+](i) oscillations of these bone cells in intact bone explants. Another possible [Ca2+](i) transient system to be considered is gap junctional communication through which Ca2+ and other messenger molecules move, at least in part, across cell-cell junctions: therefore, we also investigated the role of gap junctions in the maintenance of the autonomic [Ca2+](i) oscillations observed in the intact bone. Treatment with three distinct gap junction inhibitors, 18 alpha-glycyrrhetinic acid, oleamide, and octanol, significantly reduced the proportion of responsive osteocytes, indicating that gap junctions are important for the maintenance of [Ca2+](i) oscillations in osteocytes, but less in osteoblasts. Taken together, we found that the bone cells in intact bone explants showed autonomous [Ca2+](i) oscillations that required the release of intracellular Ca2+ stores. In addition, osteocytes specifically modulated these oscillations via cell-cell communication through gap junctions, which maintains the observed [Ca2+](i) oscillations of bone cells. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2012.01.021

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

The aim of this present study was to provide a scaffold as a tool for the investigation of the effect of mechanical stimulation on three-dimensionally cultured cells. For this purpose, we developed an artificial self-assembling peptide (SPG-178) hydrogel scaffold. The structural properties of the SPG-178 peptide were confirmed by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and transmission electron microscopy (TEM). The mechanical properties of the SPG-178 hydrogel were studied using rheology measurements. The SPG-178 peptide was able to form a stable, transparent hydrogel in a neutral pH environment In the SPG-178 hydrogel, mouse skeletal muscle cells proliferated successfully (increased by 12.4 +/- 1.5 times during 8 days of incubation; mean +/- SEM). When the scaffold was statically stretched, a rapid phosphorylation of ERK was observed (increased by 2.8 +/- 0.2 times; mean +/- SEM). These results demonstrated that the developed self-assembling peptide gel is non-cytotoxic and is a suitable tool for the investigation of the effect of mechanical stimulation on three-dimensional cell culture. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biomaterials.2011.10.049

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

The microfluidic sperm-sorting (MFSS) device is a promising advancement for assisted reproductive technology. Previously, poly(dimethylsiloxiane) and quartz MFSS devices were developed and used for intracytoplasmic sperm injection. However, these disposable devices were not clinically suitable for assisted reproduction, so a cyclo-olefin polymer MFSS (COP-MFSS) device was developed. By micromachining, two microfluidic channels with different heights and widths (chip A: 0.3 x 0.5 mm; chip B: 0.1 x 0.6 mm) were prepared. Sorted sperm concentrations were similar in both microfluidic channels. Linear-velocity distribution using the microfluidic channel of chip B was higher than that of chip A. Using confocal fluorescence microscopy, it was found that the highest number of motile spermatozoa swam across the laminar flow at the bottom of the microfluidic channel. The time required to swim across the laminar flow was longer at the bottom and top of the microfluidic channels than in the middle because of the low fluid velocity. These results experimentally demonstrated that the width of microfluidic channels should be increased in the region of laminar flow from the semen inlet to the outlet for unsorted spermatozoa to selectively recover spermatozoa with high linear velocity. (C) 2011, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.rbmo.2011.09.005

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Language：English   Publishing type：Research paper (international conference proceedings)  

Using an air-actuating device, we investigated the cellular response to mechanical stimuli (MS) in mouse blastocysts. Both MS and intracellular calcium concentration ([Ca 2+] i) were quantified based on time-resolved confocal microscopy images in the polydimethylsiloxane (PDMS) microfluidic channels by deforming a 0.1-mm membrane. [Ca 2+] i was measured in a stained mouse embryo with Fluo-4 AM using confocal fluorescence microscopy. We captured a z-series stack of sections encompassing the entire embryo. When translocation velocities of the embryo and shear stress were 40 μm/s and 0.01 dyne/cm 2, respectively, a 10% increase in the sum of fluorescent intensities (FI) was observed. When blastocysts were compressed, FI also increased in response to the applied MS. Compressive force estimated from the shape of the blastocysts was approximately 0.5-2.0 μN according to a force deformation curve for the mouse embryo. The average FI and sum of FIs increased by a factor of 1.1-1.2 times compared with those observed before MS. The increase in the sum of FI indicated that enhancement of [Ca 2+] i would be induced by these MS. © 2011 IEEE.

DOI： 10.1109/MHS.2011.6102167

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE CIRCULATION SOC  

Background: It remains unclear whether sarcolemmal BKCa channels in post-hatch chick ventricular myocytes contribute to stretch-induced extrasystoles (SIE), and whether they are stretch-activated BKCa (SAKCa) channels or a non-stretch-sensitive BKCa variant.
Methods and Results: To determine the role of sarcolemmal BKCa channels in SIE and their stretch sensitivity, an isolated 2-week-old Langendorff-perfused chick heart and mathematical simulation were used. The ventricular wall was rapidly stretched by application of a volume change pulse. As the speed of the stretch increased, the probability of SIE also significantly increased, significantly shortening the delay between SIE and the initiation of the stretch. Application of 100 nmol/L of Grammostola spatulata mechanotoxin 4, a cation-selective stretch-activated channel (SAC) blocker, significantly decreased the probability of SI E. The application of Iberiotoxin, however, a BKCa channel blocker, significantly increased the probability of SIE, suggesting that a K(+) efflux via a sarcolemmal BKCa channel reduces SIE by balancing out stretch-induced cation influx via SACs. The simulation using a cardiomyocyte model combined with a new stretch sensitivity model that considers viscoelastic intracellular force transmission showed that stretch sensitivity in BKCa channels is required to reproduce the present wet experimental results.
Conclusions: Sarcolemmal BKCa channels in post-hatch chick ventricular myocytes are SAKCa channels, and they have a suppressive effect on SIE. (Ciro J 2011; 75: 2552-2558)

DOI： 10.1253/circj.CJ-11-0486

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Microtubules are structural components of the cytoskeleton that determine cell shape, polarity, and motility in cooperation with the actin filaments. In order to determine the role of microtubules in cell alignment, human airway smooth muscle cells were exposed to cyclic uniaxial stretch. Human airway smooth muscle cells, cultured on type I collagen-coated elastic silicone membranes, were stretched uniaxially (20% in strain, 30 cycles/min) for 2 h. The population of airway smooth muscle cells which were originally oriented randomly aligned near perpendicular to the stretch axis in a time-dependent manner. However, when the cells treated with microtubule disruptors, nocodazole and colchicine, were subjected to the same cyclic uniaxial stretch, the cells failed to align. Lack of alignment was also observed for airway smooth muscle cells treated with a microtubule stabilizer, paclitaxel. To understand the intracellular mechanisms involved, we developed a computational model in which microtubule polymerization and attachment to focal adhesions were regulated by the preexisting tensile stress, pre-stress, on actin stress fibers. We demonstrate that microtubules play a central role in cell reorientation when cells experience cyclic uniaxial stretching. Our findings further suggest that cell alignment and cytoskeletal reorganization in response to cyclic stretch results from the ability of the microtubule-stress fiber assembly to maintain a homeostatic strain on the stress fiber at focal adhesions. The mechanism of stretch-induced alignment we uncovered is likely involved in various airway functions as well as in the pathophysiology of airway remodeling in asthma.

DOI： 10.1371/journal.pone.0026384

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER TOKYO  

Loose fragments in spontaneous osteonecrosis of the knee (SONK) are usually removed by surgical treatment. However, the healing potential of osteonecrotic loose fragments and their clinical availability, for example as a cell source for cartilage repair and tissue engineering, have not been investigated. The objective of this study was to evaluate the cellular proliferation and redifferentiation ability of loose fragment chondrocytes for the treatment of SONK.
Cells were obtained from the remaining cartilage of chondral loose fragments or fibrocartilaginous tissue under the affected femoral condyle in SONK. The proliferation activity of loose fragment-derived chondrocytes and condyle-derived fibrochondrocytes was evaluated. In-vitro differentiation ability was assessed by PCR and histological analysis.
The deposition of proteoglycans and type II collagen were maintained in loose fragments. However, loose fragment-derived chondrocytes had lower proliferating activity than condyle-derived fibrochondrocytes. Chondrogenic redifferentiation ability was lower in loose fragment chondrocytes than in condyle fibrochondrocytes. Differentiation towards adipogenic and osteogenic lineages was not observed in loose fragment chondrocytes. On the other hand, lipid vacuoles were detected in fibrochondrocytes after adipogenic treatment.
This study demonstrated that loose fragment-derived chondrocytes in SONK had lower potential than fibrochondrocytes in cellular proliferation and redifferentiation. Our experimental results suggest that osteonecrotic loose fragments might have restricted cellular properties in the healing of SONK-related osteochondral defects.

DOI： 10.1007/s00776-011-0128-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：W B SAUNDERS CO LTD  

Objective: To investigate the mechanism of mechanical stress-induced expression and regulation of aggrecanases and examine the role of runt-related transcription factor 2 (RUNX-2) in chondrocyte-like cells.
Methods: SW1353 cells were seeded onto stretch chambers at a concentration of 5 x 10(4) cells/chamber, and a uni-axial cyclic tensile strain (CTS) (0.5 Hz, 10% stretch) was applied for 30 min. Total RNA was extracted, reverse transcribed, and analyzed by polymerase chain reaction (PCR) and real-time PCR. RUNX-2 overexpression and small interfering RNA (siRNA) targeting RUNX-2 were used to investigate the role of RUNX-2 in CTS-induced gene expression. The involvement of diverse mitogen-activated protein kinase (MAPK) pathways in the activation of RUNX-2, MMP-13 and ADAMTS-5 during CTS was examined by Western blotting.
Results: CTS induced expression of RUNX-2, MMP-13, ADAMTS-4, -5, and -9. Overexpression of RUNX-2 up-regulated expression of MMP-13 and ADAMTS-5, whereas RUNX-2 siRNA resulted in significant down-regulation of mechanically-induced MMP-13 and ADAMTS-5 expression. CTS induced activation of p38 MAPK, and CTS induction of RUNX-2. MMP-13 and ADAMTS-5 mRNA was down-regulated by the selective p38 MAPK inhibitor SB203580 but not by the p44/42 MAPK inhibitor U0126, or the JNK MAPK inhibitor JNK inhibitor II.
Conclusions: RUNX-2 might have a role as a key downstream mediator of p38&apos;s ability to regulate mechanical stress-induced MMP-13 and ADAMTS-5 expression. (C) 2010 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.joca.2010.11.004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Institute of Electrical Engineers of Japan  

Our group previously reported the application of a flow-through type pH/CO2 sensor system designed to evaluate the metabolic activity of cultured cells. The sensor system consists of two ion-sensitive field effect transistors (ISFETs), an ISFET to measure the total pH change and an ISFET enclosed within a gas-permeable silicone tube to measure the pH change attributable to CO2. In that study, we used the system to quantitatively analyze metabolic switching induced by glucose concentration changes in three cultured cell types (bovine arterial endothelium cell (BAEC), human umbilical vein endothelium cell (HUVEC), and rat cardiomuscle cell (RCMC)), and to measure the production rates of total carbonate and free lactic acid in the cultured cells. In every cell type examined, a decrease in the glucose concentration led to an increase in total carbonate, a product of cellular respiration, and a decrease of free lactic acid, a product of glycolysis. There were very significant differences among the cell types, however, in the glucose concentrations at the metabolic switching points. We postulated that the cell has a unique switching point on the metabolic pathway from glycolysis to respiration. In this paper we use our sensor system to evaluate the metabolic switching of human embryonic kidney 293 cells triggered by glucose concentration changes. The superior metabolic pathway switched from glycolysis to respiration when the glucose concentration decreased to about 2 mM. This result was very similar to that obtained in our earlier experiments on HUVECs, but far different from our results on the other two cells types, BAECs and RCMCs. This sensor system will be useful for analyzing cellular metabolism for many applications and will yield novel information on different cell types. © 2011 The Institute of Electrical Engineers of Japan.

DOI： 10.1541/ieejeiss.131.1535

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.51.S26_3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Aminopeptidase A (APA; EC 3.4.11.7) is a transmembrane metalloprotease with several functions in tumor angiogenesis. To investigate the role of APA in the process of ischemia-induced angiogenesis, we evaluated the cellular angiogenic responses under hypoxic conditions and the process of perfusion recovery in the hindlimb ischemia model of APA-deficient (APA-KO; C57B16/J strain) mice.
Western blotting of endothelial cells (ECs) isolated from the aorta of APA-KO mice revealed that the accumulation of hypoxia-inducible factor-1 alpha (HIF-1 alpha) protein in response to hypoxic challenge was blunted. Regarding the proteasomal ubiquitination, a proteasome inhibitor MG-132 restored the reduced accumulation of HIF-1 alpha in ECs from APA-KO mice similar to control mice under hypoxic conditions. These were associated with decreased growth factor secretion and capillary formation in APA-KO mice. In the hindlimb ischemia model, perfusion recovery in APA-KO mice was decreased in accordance with a significantly lower capillary density at 2 weeks. Regarding vasculogenesis, no differences were observed in cell populations and distribution patterns between wild type and APA-KO mice in relation to endothelial progenitor cells.
Our results suggested that Ischemia-induced angiogenesis is impaired in APA-KO mice partly through decreased HIF-1 alpha stability by proteasomal degradation and subsequent suppression of HIF-1 alpha-driven target protein expression such as growth factors. APA is a functional target for ischemia-induced angiogenesis. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2010.10.043

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOCIETY REPRODUCTION & DEVELOPMENT-SRD  

Under physiological conditions, mammalian oocytes and embryos appear to be stimulated not only chemically but also mechanically, such as by compression, shear stress and/or friction force in the follicle and female reproductive tract. The present study was undertaken to examine the effects of kinetic culture with a tilting device in chemically defined media during in vitro maturation (IVM) of porcine oocytes and in vitro culture (IVC) following in vitro fertilization (IVF) on the early developmental competence and quality of blastocysts. After culture in a chemically defined IVM medium, modified porcine oocyte medium (mPOM) containing gonadotropins and dibutyryl cAMP for 20 h, the mean diameter of the cumulus-oocyte complexes (COCs) was larger in the tilting culture than in the static controls, whereas the diameter of the oocytes did not differ. When culture of the COCs was continued additionally in a fresh medium without gonadotropins and dibutyryl cAMP for 24 h, the incidences of oocytes completing GVBD and developing to the metaphase-II stage did not differ between the tilting and static culture systems. Furthermore, the sperm penetration after IVF and developmental competence of the oocytes to the blastocyst stage were not different between the tilting and static systems during IVM and IVC. However, tilting culture during both IVM and IVC had a significant positive effect on the number of cells per blastocyst (P&lt;0.05). These observations indicate that tilting culture during IVM and IVC in chemically defined media improves the quality of blastocyst, as determined by the number of cells per blastocyst, without any effects on penetrability and developmental competence.

DOI： 10.1262/jrd.10-041H

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

The objective of this study was to use a microfluidic sperm sorter (MFSS), designed to isolate motile human spermatozoa with laminar flows (no centrifugation), for porcine IVF. Boar spermatozoa were diluted at 1 x 10(8) with a diluent containing 20% seminal fluid and flowed with modified TCM-199 (mM199, with 5 mM caffeine) to introduce motile sperm into the exit chamber for IVF. In Experiment 1, after flowing for 5 min, sperm concentration varied significantly among specific sites within the MFSS collecting chamber (range, 0.8 +/- 0.5 x 10(4) to 575.0 +/- 56.3 x 10(4) cells/mL; mean +/- SEM). In Experiment 2, when porcine IVM oocytes were placed at three locations in the MFSS exit chamber (where only motile spermatozoa accumulated) and subsequently cultured in caffeine-free mM199 for 8 h, sperm penetration rate was not significantly different among places (86.1 +/- 10.5 to 100%), but the monospermic penetration rate was lower (P &lt; 0.05) in oocytes 3.5 mm from the exit position (12.5 +/- 4.8%) than those at 7.5 mm (53.1 +/- 6.0%) or further (41.9 +/- 2.8%) from the exit. In Experiment 3, the normal fertilization index (ratio of monospermic oocytes to number of oocytes examined) 8 h after insemination was higher (P &lt; 0.05) in the MFSS-IVF system (0.375 +/- 0.040) than both standard IVF and transient IVF (0.222 +/- 0.028 and 0.189 +/- 0.027, respectively, with co-culture for 8 h and for 5 min). Developmental competence of fertilized oocytes (blastocyst formation) was higher (P &lt; 0.05) in the MFSS-IVF system (40.9 +/- 2.3%) than in either standard or transient IVF (22.6 +/- 1.4 and 33.7 +/- 3.5%). In conclusion, brief co-culture of porcine oocytes with spermatozoa gradually accumulated in the MFSS chamber improved the efficiency of producing monospermic fertilized embryos and blastocysts. Furthermore, efficiencies were significantly affected by oocyte location within the chamber. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.theriogenology.2010.04.011

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Language：Japanese   Publisher：(公社)日本矯正歯科学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Blastocyst quality score (BQS), first reported by Rehman et al., is a numerical blastocyst-morphology grading system based on the criteria established by Gardner and Schoolcraft. We demonstrate a positive correlation between the calculated BQS score and cell number by staining thawed human embryos and suggest that BQS can be applied to evaluate culture systems clinically. (Fertil Steril (R) 2010;94:1135-7. (C)2010 by American Society for Reproductive Medicine.)

DOI： 10.1016/j.fertnstert.2009.11.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER THORACIC SOC  

During high tidal volume mechanical ventilation in patients with acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), regions of the lung are exposed to excessive stretch, causing inflammatory responses and further lung damage. In this study, the effects of mechanical stretch on intracellular Ca(2+) concentration ([Ca(2+)](i)), which regulates a variety of endothelial properties, were investigated in human pulmonary microvascular endothelial cells (HPMVECs). HPMVECs grown on fibronectin-coated silicon chambers were exposed to uniaxial stretching, using a cell-stretching apparatus. After stretching and subsequent unloading, [Ca(2+)](i), as measured by fura-2 fluorescence, was transiently increased in a strain amplitude-dependent manner. The elevation of [Ca(2+)](i) induced by stretch was not evident in the Ca(2+)-free solution and was blocked by Gd(3+), a stretch-activated channel inhibitor, or ruthenium red, a transient receptor potential vanilloid inhibitor. The disruption of actin polymerization with cytochalasin D inhibited the stretch-induced elevation of [Ca(2+)](i). In contrast, increases in [Ca(2+)](i) induced by thapsigargin or thrombin were not affected by cytochalasin D. Increased actin polymerization with sphingosine-1-phosphate or jasplakinolide enhanced the stretch-induced elevation of [Ca(2+)](i). A simple network model of the cytoskeleton was also developed in support of the notion that actin stress fibers are required for efficient force transmission to open stretch-activated Ca(2+) channels. In conclusion, mechanical stretch activates Ca(2+) influx via stretch-activated channels which are tightly regulated by the actin cytoskeleton different from other Ca(2+) influx pathways such as receptor-operated and store-operated Ca(2+) entries in HPMVECs. These results suggest that abnormal Ca(2+) homeostasis because of excessive mechanical stretch during mechanical ventilation may play a role in the progression of ALI/ARDS.

DOI： 10.1165/rcmb.2009-0073OC

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

We have previously reported the electrophysiological properties of sarcolemmal stretch-activated BKCa (SAKCA) channels cloned from cultured chick embryonic ventricular myocytes. However, the role of BKCa channels in the electrophysiology of the more mature heart is not clear. We have investigated the effects on the BKCa current of axial stretch in post-hatch ventricular myocytes. Whole-cell currents of ventricular myocytes isolated from 2-week-old chicks were recorded using the patch-clamp technique, while the cells were either held at resting length or stretched to cause a 10% increase in sarcomere length using a pair of carbon fibres attached to opposite ends of the cell. Stretch did not affect whole-cell currents immediately after the stretch was applied. However, sustained stretch for 3 min significantly increased outward currents. This stretch-induced change was reversed by applying 10 nm iberiotoxin, a specific BKCa channel blocker, or a Na+-Ca2+-free environment. These results were reproduced in a computer simulation study. The present study is the first report about the sarcolemmal BKCa current from post-hatch ventricular myocytes. The present results suggest that axial stretch activates BKCa channels via a stretch-induced increase in the cytosolic Na+ concentration followed by an increased Ca2+ influx.

DOI： 10.1113/expphysiol.2009.051896

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：REPRODUCTIVE HEALTHCARE LTD  

Mammalian embryos experience not only hormonal but also mechanical stimuli, such as shear stress, compression and friction force in the Fallopian tube before nidation. In order to apply mechanical stimuli to embryos in a conventional IVF culture system, the tilting embryo culture system (TECS) was developed. The observed embryo images from the TECS suggest that the velocities and shear stresses of TECS embryos are similar to those experienced in the oviduct. Use of TECS enhanced the development rate to the blastocyst stage and significantly increased the cell number of mouse blastocysts (P &lt; 0.05). Although not statistically significant, human thawed embryos showed slight improvement in development to the blastocyst stage following culture in TECS compared with static controls. Rates of blastocyst formation following culture in TECS were significantly improved in low-quality embryos and those embryos cultured under suboptimal conditions (P &lt; 0.05). The TECS is proposed as a promising approach to improve embryo development and blastocyst formation by exposing embryos to mechanical stimuli similar to those in the Fallopian tube. (C) 2009, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.rbmo.2009.12.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE SA  

The pH values of Solutions have been commonly measured by glass electrodes, devices which respond on a time scale of minutes and require complicated operations Such as calibration at every measurement. The development of a fast, automated pH measurement system for analytes in small volumes would be quite useful in many fields. Yet the practical application of such a device has been hampered by the need for rapid measurement and precise calibration. Our group has constructed a fully automated, self-calibrating pH measurement system capable of consecutively measuring 96 samples. The system uses a flow-through-type differential pH sensor probe based on a tiny, quickly responding pH-sensitive field-effect transistor (pH-FET) technology. The system was confirmed to measure the pH values of 26 mu l solutions over a 135 s cycle (60 s for wash, 20 s for movement, 3 s for Suction, and 52 s for measurement). The measurement error Of multiple sample measurements, including measurements of solutions with low buffering capacity, was -0.0116 +/- 0.0481 pH (average error +/- S.D.). The high accuracy and high reproducibility of the results Suggest that this automatic pH measurement system based on pH-FET can be applied for versatile uses. (C) 2009 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.snb.2009.09.045

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Language：English   Publishing type：Research paper (international conference proceedings)  

Throughput in the cell response analysis is expected to progress in medicine and biology. In this paper, we construct a method for the measurement of cell activity that is measured by the pH fluctuation of the solution around the cell. To improve throughput of the measurements, first we constructed an automatic pH measurement system using ion-sensitive field-effect transistor (ISFET), and the system composed of 96-well microplate, pump, XYZ stage, and PC. The basic data of the cell activity was taken from human embryonic kidney 293 (HEK 293) cells by the use of flow-through type measuring chamber. ©2010 IEEE.

DOI： 10.1109/MHS.2010.5669583

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Language：English   Publishing type：Research paper (international conference proceedings)  

Mammalian embryos experience not only hormonal but also mechanical stimuli, such as shear stress, compression, and friction force, in the fallopian tube before nidation. We aim to develop a novel and simple system to apply mechanical stimuli (MS) similar to those generated inside the oviduct to cultured mammalian embryos. Possible MS include shear stress (SS) caused by fluid dynamics and compression of embryos due to interactions with the wall of the oviduct. A new culture system was developed to increase SS and to apply MS during in vitro embryo cultures. We developed an air actuating system with microfluidic channels to apply MS by deforming a 0.1-mm-thick poly(dimethylsiloxiane) membrane and evaluated MS applied to ICR mouse embryos inside the microfluidic channel. Using this air actuating system, we applied compression to mouse embryos inside the medium channel and estimated SS on the basis of the velocity of the embryos' motion. By changing the syringe velocity, we applied different types of MS to the em bryos. These results suggested that multiple MS such as SS and compression can be applied at the same time. MS applied using this system was similar to those generated in the physiological environment of the oviduct. ©2010 IEEE.

DOI： 10.1109/MHS.2010.5669581

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.20965/jrm.2010.p0197

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：IEEE-INST ELECTRICAL ELECTRONICS ENGINEERS INC  

Analysis of cellular responses to chemicals at high spatiotemporal resolution is required for precise understanding of intracellular signal transduction. Here, we demonstrated a novel method for applying different solutions to a part of or all of a cell at high spatiotemporal resolution. We fabricated a microfluidic device using polydimethylsiloxane, and the sharp interface between the two solution streams flowing in the channel was used for the application of different solutions. We constructed a computer-controlled system to control the interface movement precisely, rapidly, and reproducibly during positioning, and spatial and temporal resolutions attained were 1.6 mu m and 189 ms, respectively. We then applied the present system to the analysis of intracellular responses to chemicals. We were able to measure [Ca(2+)](i) increases within 500 ms, when one laminar stream covered a part of the cell. This method can be used as a generic platform to investigate responses against drugs at the single cell level.

DOI： 10.1109/TNB.2009.2035253

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

In patients with acute respiratory distress syndrome, mechanical over-distension of the lung by a large tidal volume causes further damage and inflammation, called ventilator-induced lung injury (VILI), however, it is unclear how mechanical stretch affects the cellular functions or morphology in human pulmonary microvascular endothelial cells (HPMVECs). IL-8 has been proposed to play an important role in the progression of VILI by activating neutrophils. We demonstrated that HPMVECs exposed to cyclic uni-axial stretch produce IL-8 protein with p38 activation in strain- and time-dependent manners. The IL-8 synthesis was not regulated by other signal transduction pathways such as ERK1/2, JNK, or stretch-activated Ca(2+) channels. Moreover, cyclic stretch enhanced IL-6 and monocyte chemoattractant protein-1 production and reoriented cell perpendicularly to the stretch axis accompanied by actin polymerization. Taken together, IL-8 production by HPMVECs due to excessive mechanical stretch may activate neutrophilic inflammation, which leads to VILI. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2009.09.020

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Objective: Bone marrow cell therapy contributes to collateral formation through the secretion of angiogenic factors by progenitor cells and muscle cells per se, thereby presenting a novel option for patients with critical limb ischemia. However, some cases are refractory to this therapy due to graft failure. Therefore, we used genetic modification of mesenchymal stem cells (MSCs) to overexpress a vasoregulatory protein, prostacyclin (PGI(2)), to examine whether it could enhance engraftment and neovascularization in hindlimb ischemia.
Methods and results: We engineered the overexpression of PGI(2) synthase (PGIS) within MSCs, which resulted in higher expression levels of phosphorylated Akt and Bcl-2 than in control. Under hypoxic conditions. the overexpression of PGIS led to upregulated expression of cyclooxigenase-2 and peroxisome proliferator-activated receptor 8, following a 40% increased rate of proliferation in MSCs. We then produced unilateral hindlimb ischemia in C57BL6/J mice, which were injected either with MSCs transfected with GFP, with MSCs overexpressing PGIS, or with vehicle. Laser Doppler analyses demonstrated that the administration of MSCs effectively recovered blood perfusion, and that the peak blood flow was reached within 7 days of surgery in mice with MSCs overexpressing PGIS, which was earlier than that in mice with MSCs transfected with GFP. This beneficial effect was correlated to enhanced collateral formation and muscle bundle proliferation.
Conclusion: Sustained release of PGI(2) enhanced the proangiogenic function of MSCs and subsequent muscle cell regrowth in the ischemic tissue suggesting potential therapeutic benefits of cell-based gene therapy for critical limb ischemia. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.atherosclerosis.2009.02.023

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

Biomechanical stimuli have fundamental roles in the maintenance and remodeling of ligaments including collagen gene expressions. Mechanical stretching signals are mainly transduced by cell adhesion molecules such as integrins. However. the relationships between stress-induced collagen expressions and integrin-mediated cellular behaviors are still unclear in anterior cruciate ligament cells. Here, we focused on the stretch-related responses of different cells derived from the ligament-to-bone interface and midsubstance regions of human anterior cruciate ligaments. Chondroblastic interface cells easily lost their potential to produce collagen genes in non-stretched conditions, rather than fibroblastic midsubstance cells. Uni-axial mechanical stretches increased the type I collagen gene expression of interface and midsubstance cells up to 14- and 6-fold levels of each non-stretched control, respectively. Mechanical stretches also activated the stress fiber formation by shifting the distribution of integrin alpha V beta 3 to the peripheral edges in both interface and midsubstance cells. In addition, integrin alpha V beta 3 colocalized with phosphorylated focal adhesion kinase in stretched cells. Functional blocking analyses using anti-integrin antibodies revealed that the stretch-activated collagen gene expressions on fibronectin were dependent on integrin alpha V beta 3-mediated cellular adhesions in the interface and midsubstance cells. These findings suggest that the integrin alpha V beta 3-mediated stretch signal transduction might have a key role to stimulate collagen gene expression in human anterior cruciate ligament, especially in the ligament-to-bone interface. (C) 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jbiomech.2009.06.016

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

We previously reported that SAK(CA), a stretch-activated, large-conductance, calcium- and voltage-activated potassium (BKCa) channel is present in chick embryonic heart. Here, we cloned SAK(CA) and identified that Stress-Axis Regulated Exon (STREX) is responsible for the stretch sensitivity. Single patch-clamp recordings from CHO cells transfected with the cloned SAKCA showed stretch sensitivity, whereas deletion of the STREX insert diminished the stretch sensitivity of the channel. Sequence analysis revealed that the ERA(672-674) sequence of the STREX is indispensable for channel stretch sensitivity and single amino acid substitution from Ala674 to Thr674 completely eliminated the stretch sensitivity. Co-expression of chick STREX-EGFP and SAK(CA) in CHO cells, induced a strong GFP signal in the cell membrane and inhibited the stretch sensitivity significantly. These results suggest that SAK(CA) senses membrane tension through an interaction between STREX and submembranous components. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2009.05.105

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Transient receptor potential channels have recently been implicated in physiological functions in a urogenital system. In this study, we investigated the role of transient receptor potential vanilloid 4 (TRPV4) channels in a stretch sensing mechanism in mouse primary urothelial cell cultures. The selective TRPV4 agonist, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD) evoked Ca2+ influx in wild-type (WT) urothelial cells, but not in TRPV4-deficient (TRPV4KO) cells. We established a cell-stretch system to investigate stretch-evoked changes in intracellular Ca2+ concentration and ATP release. Stretch stimulation evoked intracellular Ca2+ increases in a stretch speed- and distance-dependent manner in WT and TRPV4KO cells. In TRPV4KO urothelial cells, however, the intracellular Ca2+ increase in response to stretch stimulation was significantly attenuated compared with that in WT cells. Stretch-evoked Ca2+ increases in WT urothelium were partially reduced in the presence of ruthenium red, a broad TRP channel blocker, whereas that in TRPV4KO cells did not show such reduction. Potent ATP release occurred following stretch stimulation or 4 alpha-PDD administration in WT urothelial cells, which was dramatically suppressed in TRPV4KO cells. Stretch-dependent ATP release was almost completely eliminated in the presence of ruthenium red or in the absence of extracellular Ca2+. These results suggest that TRPV4 senses distension of the bladder urothelium, which is converted to an ATP signal in the micturition reflex pathway during urine storage.

DOI： 10.1074/jbc.M109.020206

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

This paper describes a study in which numerical simulations were applied to improve the separation efficiency of a microfluidic-based sperm sorter. Initially, the motion of 31 sperm were modeled as a sinusoidal wave. The modeled sperm were expected to move while vibrating in the fluid within the microchannel. In this analysis, the number of sperm extracted at the outlet channel and the rate of movement of the highly motile sperm were obtained for a wide range of flow velocities within the microchannel. By varying the channel height, and the width and the position of the sperm-inlet channel, we confirmed that the separation efficiency was highly dependent on the fluid velocity within the channel. These results will be valuable for improving the device configuration, and might help to realize further improvements in efficiency in the future.

DOI： 10.1007/s10544-008-9207-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：GEORG THIEME VERLAG KG  

Insulin-like growth factor I (IGF-I), an autocrine/paracrine growth factor involved in myogenesis, has rapid effects on muscle metabolism. In a manner analogous to insulin and mechanical stimuli such as stretch, IGIF-I stimulates glucose transport through recruitment of glucose transporters to surface membranes in skeletal muscles. It is known that IGF-I is secreted from skeletal muscle cells in response to stretch. Therefore, we examined whether IGF-I is involved in the mechanism by which mechanical stretch regulates glucose transport using cultured C2C12 myotubes. IGF-I increased 2-deoxy-D-glucose (2-DG) uptake, and this created an additive effect with mechanical stretch, suggesting that these stimuli enhance glucose transport through different mechanisms. In fact, IGF-I-stimulated 2-DG uptake was not blocked by dantrolene (an inhibitor of Ca2+ release from sarcoplasmic reticulum), whereas the stretch-stimulated effect was abolished. Conversely, the IGF-I-stimulated 2-DG uptake was prevented by phosphatidylinositol 3-kinase inhibitor wortmannin, which did not prevent the stretch-stimulated 2-DG uptake. In addition, experiments using media conditioned by stretched myotubes indicated that a mechanically induced release of locally acting autocrine/paracrine growth factors was not sufficient for induction of 2-DG uptake. Thus, our results demonstrate that mechanical stretch signaling for glucose transport is independent of the mechanism through which IGF-I increases this transport.

DOI： 10.1055/s-0028-1087170

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Polymeric curved structures are widely used in imaging systems including optical fibers and microfluidic channels. Here, we demonstrate that small-scale, poly(dimethylsiloxane) (PDMS)-based, curved structures can be fabricated through controlling interfacial free energy equilibrium. Resultant structures have a smooth, symmetric, curved surface, and may be convex or concave in form based on surface tension balance. Their curvatures are controlled by surface characteristics (i.e., hydrophobicity and hydrophilicity) of the molds and semi-liquid PDMS. In addition, these structures are shown to be biocompatible for cell culture. Our system provides a simple, efficient and economical method for generating integrateable optical components without costly fabrication facilities.

DOI： 10.1039/b910826b

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：IOS PRESS  

Impaired deformability might contribute to the accumulation of activated leukocytes within pulmonary microcapillaries, leading to acute lung injury. The purpose of our study was to investigate changes in leukocyte deformability during periods of inflammation after esophagectomy. The study group comprised 20 patients who underwent esophagectomy. Changes in leukocyte deformability were investigated by examining filtration through a silicon microchannel, which simulated human pulmonary microcapillaries. Changes in the neutrophil cytoskeleton were investigated by measuring neutrophil F-actin assembly. The severity of patient clinical outcome was evaluated by the lung injury score. Leukocyte filtration through the microchannel was significantly weaker in esophagectomy patients than in healthy subjects (p &lt; 0.01). After esophagectomy, filtration was further impaired compared with preoperative values (p &lt; 0.05). The neutrophil F-actin content was higher in patients than in controls (p &lt; 0.01), and increased after esophagectomy compared with preoperative values (p &lt; 0.01). We concluded that circulating leukocytes showed reduced deformability and appeared to be sequestered within microcapillaries after esophagectomy. Changes in neutrophil cytoskeleton were considered to be responsible for the reduced deformability. Leukocyte accumulation within pulmonary microcapillaries might be related to the pathogenesis of lung injury after esophagectomy.

DOI： 10.3233/CH-2009-1164

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE SA  

We have previously reported that a flow-through type pH/CO2 sensor system using two ion sensitive field effect transistors (ISFETs) could be utilized to evaluate the production rate of total carbonate (secreted CO2 + secreted bicarbonate ion) and free lactic acid (secreted lactic acid -secreted bicarbonate ion). To validate the usefulness of this system, we applied it to the quantitative analysis of metabolic switching by the change Of glucose concentrations in cultured bovine arterial endothelium cell, human umbilical vein endothelium cell and rat cardiac muscle cell. In all of these cell species, a decrease of glucose concentration increased total carbonate that represents the cellular respiration activity and decreased free lactic acid that represents glycolysis activity. We firstly analyzed a switching of metabolic pathways from glycolysis to respiration with a decrease of glucose quantitatively. This ISFET system can be applicable to many biological investigations by analyzing metabolic activity and give dynamic cellular information. (C) 2008 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.snb.2008.05.021

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Background and purpose: In the ciliary muscle, the tonic component of the contraction produced by cholinergic agonists is highly dependent on Ca(2+) provided by influx through non-selective cation channels (NSCCs) opened by stimulation of M(3) muscarinic receptors. We examined effects of YM-254890 (YM), a G(q/11)-specific inhibitor, on contraction, NSCC currents and [Ca(2+)](i) elevation induced by carbachol (CCh).
Experimental approach: Isometric tension was recorded from ciliary muscle bundles excised from bovine eyes. In ciliary myocytes dispersed with collagenase and cultured for 1-5 days, whole-cell currents were recorded by voltage clamp and the intracellular free Ca(2+) concentration [Ca(2+)](i) was monitored using the Fluo-4 fluorophore. Existence and localization of M(3) receptors and the a subunit of G(q/11) (G alpha(q/11)) were examined by immunofluorescence microscopy using AlexaFluor-conjugated antibodies.
Key results: Both phasic and tonic components of contractions evoked by 2 mu M CCh were inhibited by YM (3-10 mu M) in a dose-dependent manner. In the cultured cells, CCh (0.05-10 mu M) evoked an NSCC current as well as an elevation of the [Ca(2+)](i). Both initial and sustained phases of these CCh-evoked responses were abolished by YM (3-10 mu M). Immunostaining of the cytoplasmic side of the plasma membrane of ciliary myocytes revealed a dense distribution of M(3) receptors and G alpha(q/11).
Conclusions and implications: The tonic as well as phasic component of the ciliary muscle contraction appears to be under control of signals conveyed by a G(q/11)-coupled pathway. YM is a useful tool to assess whether G(q/11) is involved in a signal transduction system.

DOI： 10.1038/bjp.2008.140

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER THORACIC SOC  

In response to mechanical stretch, airway smooth muscle exhibits various cellular functions such as contraction, proliferation, and cytoskeletal remodeling, all of which are implicated in the pathophysiology of asthma. We tested the hypothesis that mechanical stretch of airway smooth muscle cells increases intracellular Ca2+ concentration ([Ca2+](i)) by activating stretch-activated (SA) nonselective cation channels. A single uniaxial stretch (3 s) was given to human bronchial smooth muscle cells cultured on an elastic silicone membrane. After the mechanical stretch, a transient increase in [Ca2+], was observed. The [Ca2+](i) increase was significantly dependent on stretch amplitude. The augmented [Ca2+](i) due to stretch was completely abolished by removal of extracellular Ca2+ and was markedly attenuated by an application of Gd3+, an inhibitor of SA channels, or ruthenium red, a transient receptor potential vanilloid (TRPV) inhibitor. In contrast, the stretch-induced rises of [Ca2+](i) were not altered by other Ca2+ channel inhibitors such as nifedipine, BTP-2, and SKF-96365. Moreover, the [Ca2+](i) increases were not affected by indomethacin, a cyclooxygenase inhibitor, U-73122, a phospholipase C inhibitor, or xestospongin C, an inhibitor of the inositol-trisphosphate receptor. These findings demonstrate that a novel Ca2+ influx pathway activated by mechanical stretch, possibly through the Ca2+-permeable SA channel activated directly by stretch rather than by indirect mechanisms via intracellular messenger production, is involved in human airway smooth muscle cells. A molecular candidate for the putative SA channel may be one of the members of the TRPV channel family. Thus, abnormal Ca2+ homeostasis in response to excessive mechanical strain would contribute to the pathogenesis of asthma.

DOI： 10.1165/rcmb.2007-0259OC

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER PHYSIOLOGICAL SOC  

Embryonic stem (ES) cells are exposed to fluid-mechanical forces, such as cyclic strain and shear stress, during the process of embryonic development but much remains to be elucidated concerning the role of fluid- mechanical forces in ES cell differentiation. Here, we show that cyclic strain induces vascular smooth muscle cell (VSMC) differentiation in murine ES cells. Flk-1-positive (Flk-1(+)) ES cells seeded on flexible silicone membranes were subjected to controlled levels of cyclic strain and examined for changes in cell proliferation and expression of various cell lineage markers. When exposed to cyclic strain (4-12% strain, 1 Hz, 24 h), the Flk-1(+) ES cells significantly increased in cell number and became oriented perpendicular to the direction of strain. There were dose-dependent increases in the VSMC markers smooth muscle alpha-actin and smooth muscle-myosin heavy chain at both the protein and gene expression level in response to cyclic strain, whereas expression of the vascular endothelial cell marker Flk-1 decreased, and there were no changes in the other endothelial cell markers (Flt-1, VE-cadherin, and platelet endothelial cell adhesion molecule 1), the blood cell marker CD3, or the epithelial marker keratin. The PDGF receptor beta(PDGFR beta) kinase inhibitor AG-1296 completely blocked the cyclic strain-induced increase in cell number and VSMC marker expression. Cyclic strain immediately caused phosphorylation of PDGFR beta in a dose-dependent manner, but neutralizing antibody against PDGF-BB did not block the PDGFR beta phosphorylation. These results suggest that cyclic strain activates PDGFR beta in a ligand-independent manner and that the activation plays a critical role in VSMC differentiation from Flk-1(+) ES cells.

DOI： 10.1152/japplphysiol.00870.2007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Tensile and compressive strains are commonly used in mechanobiological models. Here we report on the development of a novel three-dimensional cell-culture method, which allows both tensile and compressive loads to be applied. Preliminary results were obtained using HCS2/8 chondrocytic cells embedded in type I collagen gel. This construct was subjected to either 16% tension or 14% compression. Confocal laser scanning microscopy showed that both tension and compression caused significant cell deformation. The collagen gel-embedded HCS2/8 cells were subjected to static tension, dynamic tension, static compression or dynamic compression for 24 h. Dynamic compression led to significantly decreased 5-bromo-2'-deoxyuridine incorporation compared with the control group. PCR analysis revealed upregulation of type II collagen caused by dynamic tension, upregulation of aggrecan caused by static compression, and downregulation of type II collagen and aggrecan caused by dynamic compression. Nitric oxide production was significantly increased by static tension and static compression compared with the control group. Our experimental system effectively applied several types of strain to HCS2/8 cells embedded in collagen gel. Our results suggest that the mode of mechanical strain affects the response of HCS2/8 cells. (c) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jbiotee.2007.07.955

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Human vascular endothelial cells form the interface between the bloodstream and vessel walls and are continuously subjected to mechanical stimulation. When endothelial cells are stretched cyclically, along one axis, they align perpendicular to the axis of stretch. We previously reported that applying a cyclic, uni-axial strain to cells induced tyrosine phospborylation of focal adhesion kinase and stimulated mitogen-activated protein kinase. However, it is difficult to quantify and analyze the spatial distribution of tyrosine phosphorylation in these cells, as they form focal adhesions randomly. In this study, we developed a system to overcome this problem by preparing individual, uniform, patterned cells that could be stretched cyclically and uni-axially. We constructed polydimethylsiloxane stretch chambers and used microcontact printing technology to imprint a pattern of 2 mu m fibronectin dots (10 lines x 10 columns in a 38 mu m square) before seeding them with human umbilical vein endothelial cells (HUVEC). We found that most HUVEC attached to the patterned dots after 2 h and were similar in size and morphology, based on phase-contrast microscopy. In this system we were able to statistically analyze tyrosine phosphorylation and actin polymerization in these patterned cells, when subjected to a cyclic, uni-axial strain, using fluorescent microscopy. (C) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jbiotec.2007.09.017

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER PHYSIOLOGICAL SOC  

Pulmonary hypertension (PH) causes right ventricular (RV) hypertrophy and, according to the extent of pressure overload. eventual heart failure. We tested the hypothesis that the mechanical stress in PH-RV impairs the vasoreactivity of the RV coronary microvessels of different sizes with increased Superoxide levels. Five-week-old male Sprague-Dawley rats were injected with monocrotaline (n = 126) to induce PH or with saline as controls (n = 114). After 3 wk. coronary arterioles (diameter = 30-100 mu m) and small arteries (diameter =100-200 mu m) in the RV were visualized using intravital videomicroscopy. We evaluated ACh-induced vasodilation alone, in the presence of N-omega-nitro-L-arginine methyl ester (L-NAME), in the presence of tetraethylammonium (TEA) or catalase with or without L-NAME, and in the presence of SOD. The degree of suppression in vasodilation by L-NAME and TEA was used as indexes of the Contributions of endothelial nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF), respectively. In PH rats. ACh-induced vasodilation was significantly attenuated in both arterioles and small aretries. especially in arterioles. This decreased vasodilation was largely attributable to reduced NO-mediated vasoreactivity. whereas the EDHF-mediated vasodilation was relatively robust. The Suppressive effect on arteriolar vasodilation by catalase was similar to TEA in both groups. Superoxide, as measured by Lucigenin chemiluminescence, was significantly elevated in the RV tissues in PH. SOD significantly ameliorated the impairment of ACh-induced vasodilation in PH. Robust EDHF function will play a protective role in preserving coronary microvascular homeostasis in the event of NO dysfunction with increased superoxide levels.

DOI： 10.1152/ajpheart.00548.2006

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Language：English   Publisher：The Institute of Electrical Engineers of Japan  

Automatic pH measurement system was developed by using a flow-through type differential pH-FET sensor. The system was operated by repeating 60 sec cycle consisting of 36 sec of washing period, 4 sec of moving period, and 20 sec of measuring period. In washing period, the differential pH-FET sensor was washed with baseline solution. In measuring period, about 50 μl of sample solution was withdrawn to the space around a measurement pH-FET, and the change of differential source potential was measured. Absolute values of error for nine sample solutions with weak buffering ability were less than 0.146 pH, and were 0.071 pH in average. From this experiment, it was suggested that high-speed and automated pH measurement will be possible by using pH-FET sensors even for sample solutions of weak buffer ability.

DOI： 10.1541/ieejsmas.127.367

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

transport and glycogen metabolism in skeletal muscle. However, the molecular mechanisms involved in the mechano-transduction events are poorly understood. The present study was conducted in order to determine whether the signaling mechanism leading to mechanical stretch-stimulated glucose transport is similar to, or distinct from, the signaling mechanisms leading to insulin- and contraction-stimulated glucose transport in cultured muscle cells. Methods: Cultured C2C12 myotubes were stretched, after which the 2-deoxy-D -glucose (2-DG) uptake was measured. Results: Following cyclic stretch, C2C12 myotubes showed a significant increase in 2-DG uptake, and this effect was not prevented by inhibiting phosphatidylinositol 3-kinase or 5'-AMP-activated protein kinase and by extracellular Ca2+ chelation. Conversely, the stretch-stimulated 2-DG uptake was completely prevented by dantrolene (an inhibitor of Ca2+ release from sarcoplasmic reticulum). Furthermore, the stretch-stimulated 2-DG uptake was prevented by the Ca2+/calmodulin-dependent kinase inhibitor KN93 which did not prevent the insulin-stimulated 2-DG uptake. Conclusions: These results suggest that the effects of stretch-stimulated glucose transport are independent of the insulin-signaling pathway. By contrast, following mechanical stretch in skeletal muscle, the signal transduction pathway leading to glucose transport may require the participation of cytosolic Ca2+ and Ca2+/calmodulin kinase, but not 5'-AMP-activated protein kinase. Copyright (c) 2007 S. Karger AG, Basel.

DOI： 10.1159/000103375

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.47.S188_1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

In guinea pigs, it is well-known that mechanical stretch of airway smooth muscle exhibits spontaneous tone which is mediated by cyclooxygenase (COX) activation. We tested the hypothesis that this spontaneous contraction of airway smooth muscle is mediated by stretch-activated non-selective cation channels and the Rho/Rho-kinase pathway, as well as COX-2 using a pharmacological approach. Isometric force and intracellular Ca2+ concentrations ([Ca2+](i)) were assessed in isolated guinea pig tracheal smooth muscle tissues. The samples were stretched to a given level and the muscle behavior was monitored under isometric conditions. We observed an increase in [Ca2+](i) and subsequent force generation over a 15-min period. The augmented [Ca2+](i) and spontaneous contraction due to the stretch were markedly attenuated by application of Gd3+, an inhibitor of stretch-activated channels, and removal of extracellular Ca2+. In contrast, nifedipine only had a mild inhibitory effect on the contraction. (R)-(+)trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane-carboxamide (Y-27632; a Rho-kinase inhibitor) abolished the spontaneous contraction with no changes in [Ca2+](i). Simvastatin, which down-regulates Rho activity, also significantly inhibited the contraction. Moreover, indomethacin, an inhibitor of COX-1 and -2, and N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398; a COX-2 inhibitor) abolished the stretch-induced contraction without affecting [Ca2+](i), whereas the inhibitory effect of 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC560; a COX-1 inhibitor) on the contraction was much less. These findings demonstrated that Ca2+ entry via stretch-activated channels, the Rho/Rho-kinase pathway, and COX-2 are involved in the mechanotransduction in guinea pig tracheal smooth muscle. Additionally, while the Rho/Rho-kinase pathway and COX-2 regulate the spontaneous contraction independently of [Ca2+](i), COX-1 is not involved in the stretch-induced force generation. (c) 2006 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.ejphar.2006.08.067

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER PHYSIOLOGICAL SOC  

The viscoelastic and dynamic nonlinear properties of guinea pig tracheal smooth muscle tissues were investigated by measuring the storage (G') and loss (G') moduli using pseudorandom small-amplitude length oscillations between 0.12 and 3.5 Hz superimposed on static strains of either 10 or 20% of initial length. The G" and G' spectra were interpreted using a linear viscoelastic model incorporating damping (G) and stiffness (H), respectively. Both G and H were elevated following an increase in strain from 10 to 20%. There was no change in harmonic distortion (K-d), an index of dynamic nonlinearity, between 10 and 20% strains. Application of methacholine at 10% strain significantly increased G and H while it decreased Kd. Cytochalasin D, isoproterenol, and HA-1077, a Rho-kinase inhibitor, significantly decreased both G and H but increased Kd. Following cytochalasin D, G, H, and Kd were all elevated when mean strain increased from 10 to 20%. There were no changes in hysteresivity, G/H, under any condition. We conclude that not all aspects of the viscoelastic properties of tracheal smooth muscle strips are similar to those previously observed in cultured cells. We attribute these differences to the contribution of the extracellular matrix. Additionally, using a network model, we show that the dynamic nonlinear behavior, which has not been observed in cell culture, is associated with the state of the contractile stress and may derive from active polymerization within the cytoskeleton.

DOI： 10.1152/ajplung.00299.2005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

We investigated the signaling mechanism of stretch-induced NO (Nitric oxide) production in bovine arterial endothelial cells (BAECs). BAECs cultured on an elastic silicone chamber coated with fibronectin were subjected to uni-axial cyclic stretch (1 Hz, 20% in length) and the amount of produced NO was measured by a cGMP assay. NO production increased in a bi-phasic manner and peaked at 5 min and 20 min after stretch onset. Correspondingly, the activities of endothelial nitric oxide synthase (eNOS) and Akt/PKB (measured by phosphorylation at serine 1177 and serine 473, respectively), showed two peaks over time. Application of Gd3+, a potent SA channel blocker, and depletion of external Ca2+ exclusively inhibited the first peaks of eNOS and Akt activity, but exerted little effect on the second peak. On the other hand, the PI3K inhibitors, Wortmannin, LY294002, almost completely inhibited the second peak but not the first. These results suggest that up-regulation of eNOS in response to cyclic stretch was mediated by two distinct pathways, [Ca2+](i) increases via the SA channel in an early phase (partially Akt/PKB), and PI3K-Akt/PKB pathways in a late phase. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.lfs.2005.12.051

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：IEEE  

The rapid solution exchange around the adhesive single cell has recently received a great deal of attention from the viewpoint cell response analyses. We recently constructed a new system, which enables the replacement of solutions rapidly and precisely, by utilizing laminar flow technology. The sharp boundary of the adjoining two liquids (no mixing) moves around the cells, and minimum increments of the boundary shift are controlled at an interval of roughly 1.6 mu m, and the time required for this movement is 20 msec. The system can be used for the analysis of intracellular Ca2+ concentrations using a fluorescent indicator, in which the cells are thus stimulated by the rapid exchange of the solution. These results suggest that the spatial and temporal control of the solution around the adhesive single cell can thus be realized.

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.46.S273_2

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.46.S237_4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Some BK channels are activated in response to membrane stretch. However, it remains largely unknown which membrane component transmits forces to the channel and which part of the channel senses the force. Recently, we have shown that a BK channel cloned from chick heart (named SAKCa channel) is a stretch activated channel, while deletion of a 59 amino acids splice insert (STREX) located in the cytoplasmic side, abolishes its stretch-sensitivity. This finding raised a question whether stress in the bilayer is crucial for the mechanical activation of the channel. To address this question we examined the effects of membrane perturbing amphipaths on the stretch activation of the SAKCa channel and its STREX-deletion mutant. We found that both anionic amphipath trinitrophenol (TNP) and cationic amphipath chlorpromazine (CPZ) could dose-dependently activate the channel by leftward shifting the voltage activation curve when applied alone. In contrast, TNP and CPZ compensated each other's effect when applied sequentially. These results can be understood in the framework of the bilayer couple hypothesis, suggesting that stress in the plasma membrane can activate the SAKCa channel. Interestingly, the STREX-deletion mutant channel has much less sensitivity to the amphipaths, suggesting that STREX acts as an intermediate structure that can indirectly convey stress in the membrane to the gate of the SAKCa channel via an unidentified membrane associated protein(s) that can detect or transmit stress in the membrane.

DOI： 10.1080/09687860500370703

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

1 Uniaxial cyclic stretch leads to an upregulation of cyclooxygenase (COX)-2 through increases in the intracellular Ca2+ concentration via the stretch-activated (SA) channel and following nuclear factor kappa B (NF-kappa B)activation in human fibroblasts. However, the signaling mechanism as to how the elevated Ca2+ activates NF-kappa B is unknown. In this study, weexamined the involvement of reactive oxygen species (ROS) as an intermediate signal, which links the elevated Ca2+ with NF-kappa B activation.
2 4-Hydroxy-2-nonenal (HNE) was produced and modified I kappa B peaking at 2 min. The phosphorylation of I kappa B peaked at 8min. HNE modification and I kappa B phosphorylation, NF-kappa B translocation to the nucleus, and following COX-2 production were inhibited by extracellular Ca2+ removal or Gd3+ application, as well as by the antioxidants. The stretch-inducedCa(2+) increase was inhibited by extracellular Ca2+ removal, or Gd3+ application.
3 I kappa B kinase (IKK) activity peaked at 4 min, which was inhibited by extracellular Ca2+ removal, Gd3+ or the antioxidants. IKK was also HNE-modified and, similarly to I kappa B, peaked at 2 min. IKK under static conditions was activated by exogenously applied HNE at a relatively low dose (1 mu M), while it was inhibited at higher concentrations, suggesting thatHNE could be one of the candidate signals in the stretch-induced NF-kappa B activation.
4 The present study suggests that the NF-kappa B activation by cyclic stretch is mediated by the following signal cascade: SA channel activation -&gt; intracellular Ca2+ increase -&gt; production of ROS -&gt; activation of IKK -&gt; phosphorylation of I kappa B -&gt; NF-kappa B translocation to the nucleus.

DOI： 10.1038/sj.bjp.0706182

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER PHYSIOLOGICAL SOC  

We previously reported that uniaxial continuous stretch in human umbilical vein endothelial cells (HUVECs) induced interleukin-6 (IL-6) secretion via I kappa B kinase (IKK)/nuclear factor-kappa B (NF-kappa B) activation. The aim of the present study was to clarify the upstream signaling mechanism responsible for this phenomenon. Stretch-induced IKK activation and IL-6 secretion were inhibited by application of alpha(5)beta(1) integrin-inhibitory peptide (GRGDNP), phosphatidylinositol 3-kinase inhibitor (LY-294002), phospholipase C-gamma inhibitor (U-73122), or protein kinase C inhibitor (H7). Although depletion of intra- or extracellular Ca2+ pool using thapsigargin (TG) or EGTA, respectively, showed little effect, a TG-EGTA mixture significantly inhibited stretch-induced IKK activation and IL-6 secretion. An increase in the intracellular Ca2+ concentration ([Ca2+](i)) upon continuous stretch was observed even in the presence of TG, EGTA, or GRGDNP, but not in a solution containing the TG-EGTA mixture, indicating that both integrin activation and [Ca2+](i) rise are crucial factors for stretch-induced IKK activation and after IL-6 secretion in HUVECs. Furthermore, while PKC activity was inhibited by the TG-EGTA mixture, GRGDNP, LY-294002, or U-73122, PLC-gamma activity was retarded by GRGDNP or LY-294002. These results indicate that continuous stretch-induced IL-6 secretion in HUVECs depends on outside-in signaling via integrins followed by a PI3-K-PLC-gamma-PKC-IKK-NF-kappa B signaling cascade. Another crucial factor, [Ca2+](i) increase, may at least be required to activate PKC needed for NF-kappa B activation.

DOI： 10.1152/ajpcell.00314.2004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

DOI： 10.1038/nmat1365

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Previously we reported that a uni-axial cyclic stretch treatment of rat 3Y1 fibroblasts induced focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation (Wang et al., 2001) [Wang, J.G., Mlyazu, M., Matsushita, E., Sokabe, M., Naruse, K., 2001. Uni-axial cyclic stretch induces focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation. Biochem. Biophys. Res. Comm. 288, 356-361]. In the present study, we investigated whether stretchinduced MA13K activation leads to proliferation of fibroblasts. 3Y1 fibroblasts were subjected to a uni-axial cyclic stretch treatment (1 Hz, 120% in length) and the bromodeoxyuridine (BrdU) incorporation was measured to access cell proliferation. BrdU incorporation increased in a time-dependent manner and became significant within 6 hours. To investigate the involvement of FAK, we transiently expressed FAK mutants that lacked tyrosine phosphorylation site (s) (F397Y, F925Y, F397/925Y). Transient expression of wild-type FAK or mock vector did not inhibit the stretch-induced BrdU incorporation, however, the FAK mutants significantly blocked BrdU incorporation. Treatment of the cells with MAPK inhibitors, PD98059 or S13203580, blocked extracellular signal- regulated kinase (ERK) phosphorylation and p38 MAPK phosphorylation, respectively, and also blocked stretchinduced BrdU incorporation. These results suggest that the stretch-induced FAK activation followed by MAPK activation plays an important role in the stretch-induced proliferation of 3Y1 fibroblasts. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.lfs.2004.10.050

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：IEEE  

Here we present long-term patterning and response studies of neuroblasts on arrays of extracellular matrix (ECM) protein lines fabricated by cracking a polymer-supported thin film and selectively depositing proteins inside the cracks. Neuroblasts cultured for five days on the protein matrix switched between spreading and retraction upon changing the protein line width. This technique provides a novel tool to pursue further understanding of the basic mechanisms of neuroblast pathfinding on adhesion and morphology. Biomedical device design will also benefit from materials engineered with the reconfigurable protein patterns.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Objective: Prostacyclin synthase (PGIS) gene transfer have been shown to accelerate re-endothelialization and prevent neointimal formation in balloon-injured arteries. The aim of this study is to evaluate how overexpression of endogenous prostacyclin exerts those beneficial effects in atheromatous arteries. Methods: New Zealand White Rabbits fed a 0.5% cholesterol diet underwent balloon injury and Palmaz-Schatz stent implantation in the iliac arteries followed PGIS gene (pCMV-PGIS, 200 mug) delivery by the lipotransfection method via Dispatch catheter (n = 6 each). Results: One week after transfection, arterial segments of pCMV-PGIS produced higher levels of 6-keto-PGF1alpha than those of control, pCMV-LacZ (p &lt; 0.05). The levels of vascular endothelial growth factor (VEGF) expression was greater in the vessels of pCMV-PGIS than in those of pCMV-LacZ demonstrated by immunohistochemical analysis and quantitation of Western blotting (1.8-fold, p &lt; 0.05). At 2 weeks, in-stent endothelialization was significantly greater in the vessels of pCMV-PGIS than in those of pCMV-LacZ (p &lt; 0.01). The percentage of BrdU-positive nuclei in the injured arterial segments was lower in vessels of pCMV-PGIS than pCMV-LacZ (p &lt; 0.01). At 4 weeks, PGIS gene transfer reduced the neointimal area by 38% (p &lt; 0.05) and widened the lumen area by 71% (p &lt; 0.01). Conclusion: PGIS gene transfer accelerated re-endothelialization, and attenuated neointimal fort-nation after stent implantation in atheromatous rabbit arteries, at least in part, via increased production of VEGF protein. (C) 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.cardiores.2003.10.016

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER-VERLAG  

We have characterized electrophysiological and pharmacological properties of a stretch-activated BKca channel (SAKcaC) that was cloned from cultured chick ventricular myocytes (CCVM) and expressed in chinese hamster ovary cells (CHO) using the patch-clamp technique. Our results indicate that the cloned SAKcaC keeps most of the key properties of the native SAKcaC in CCVM, such as conductance, ion selectivity, pressure-, voltage- and Ca2+-dependencies. However, there was a slight difference between these channels in the effects of channel blockers, charybdotoxin (CTX) and gadolinium (Gd3+). The native SAKcaC was blocked in an all-or-none fashion characterized as the slow blockade, whereas the conductance of the cloned SAKcaC was gradually decreased with the blockers' concentration, without noticeable blocking noise. As the involvement of some auxiliary components was suspected in this difference, we cloned a BK beta-subunit from CCVM and coexpressed it with the cloned SAKcaC in CHO cells to examine its effects on the SAKcaC. Although the pharmacological properties of the cloned SAKcaC turned out to be very similar to the native one by the coexpression, it also significantly altered the key characteristics of SAKcaC, such as voltage- and Ca2+-dependencies. Therefore we concluded that the native SAKcaC in CCVM does not interact with the corresponding endogenous beta-subunit. The difference in pharmacological properties between the expressed SAKcaC in CHO and the native one in CCVM suggests that the native SAKca in CCVM is modulated by unknown auxiliary components.

DOI： 10.1007/s00232-003-0637-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Interleukin-6 (IL-6) secretion from endothelial cells (ECs) in response to mechanical stimuli plays an important role in the regenerative and inflammatory responses. The aim of this study was to determine the mechanism for the secretion of IL-6 from ECs in response to uni-axial continuous stretch. Continuous stretch induced IL-6 secretion from human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Reverse transcriptase-polymerase chain reaction (RT-PCR) amplification showed that the transcription of the IL-6 gene peaked 2 It after stretch. In vitro kinase assay of IkappaB kinase (IKKs) activity demonstrated that the activation of IKKs peaked 15 min after stretch. Two NF-kappaB inhibitors, pyrrolidine dithiocarbamanate (PDTC) and SN50, or antisense oligodeoxynucleotides for NF-kappaB p65 and p50 suppressed IL-6 mRNA expressions induced by continuous stretch. In conclusion, continuous stretch induces IL-6 secretion from ECs, most likely through sequential activation of IKKs and NF-kappaB. (C) 2003 Elsevier Inc. All rights reserved.

DOI： 10.1016/S0006-291X(03)01362-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

There are many experiments in which it would be useful to treat a part of the surface or interior of a cell with a biochemical reagent. It is difficult, however, to achieve subcellular specificity, because small molecules diffuse distances equal to the extent of the cell in seconds. This paper demonstrates experimentally, and analyzes theoretically, the use of multiple laminar fluid streams in microfluidic channels to deliver reagents to, and remove them from, cells with subcellular spatial selectivity. The technique made it possible to label different subpopulations of mitochondria fluorescently, to disrupt selected regions of the cytoskeleton chemically, to dislodge limited areas of cell-substrate adhesions enzymatically, and to observe microcompartmental endocytosis within individual cells. This technique does not require microinjection or immobilization of reagents onto nondiffusive objects; it opens a new window into cell biology.

DOI： 10.1016/S1074-5521(03)00019-X

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Background/Aims: Interleukin-6 (IL-6) is an essential early signal in liver regeneration, however, little is known about what triggers IL-6 release. Changes in portal hemodynamics after portal vein embolization (PVE) may contribute to IL-6 release, leading to regeneration of non-embolized lobe.
Methods: In 22 patients who underwent right PVE, the diameters of the left portal branches, liver volumes, and serum concentrations of IL-6, tumor necrosis factor-alpha (TNF-alpha), and hepatocyte growth factor (HGF) were measured. We then studied endothelial cells cultured on an elastic silicone membrane and subjected to continuous uni-axial stretch. Supernatant cytokine concentrations were measured.
Results: The diameters of the portal branches increased by 150% after PVE. Serum IL-6 concentrations increased within 3 h after PVE. The concentrations of TNF-alpha and HGF remained unchanged. The left lobe volume increased 2 weeks after PVE. The IL-6 concentrations in the supernatant of endothelial cells with stretch stress were higher than that in the non-stretched control group.
Conclusions: These findings indicate that PVE dilates the portal branches in the non-embolized lobe, exposing hepatic vasculature to stretch stress. This hemodynamic change may act as a trigger for IL-6 release from endothelial cells and contribute to the activation of regenerative cascade in the non-embolized lobes. (C) 2002 European Association for the Study of Liver. Published by Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0168-8278(02)00171-X

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

Previous studies have shown that prostacyclin (PGI(2)) synthase (PCS) gene transfer inhibits neointimal formation in balloon-injured arteries. However, the role of each cyclooxygenase (COX) isoform in this healing mechanism remains unknown. We hypothesized that overexpression of PCS may modulate COX-2-mediated prostaglandin (PG) metabolism. That is to say, excessive PGH(2) derived from COX-2 after balloon injury may be converted into PGI(2) rather than PGE(2) or thromboxane (TX) A(2) by overexpressed PCS. We examined the expression of COX isoforms and evaluated the role of COX-2 with regard to the effects of PCS gene transfer by using 4-(4-cyclohexyl-2-methyloxazol-5-yl)-2-fluorobenzenesulfonamide (JTE-522), a selective COX-2 inhibitor. Rats were divided into 4 groups in conjunction with PCS gene transfer and JTE-522 treatment. The PCS gene (30 mug) was transfected into rat balloon-injured arteries by a lipotransfection method. JTE-522 (30 mg/kg per day) was administered for 14 days after balloon injury. Immunohistochemical analysis demonstrated marked COX-2 expression on the neointima. PCS gene transfer markedly inhibited neointimal formation, but JTE-522 reversed this beneficial effect. PCS gene transfer augmented PGI(2) production and decreased PGE(2) production without affecting TXA(2) production, but JTE-522 inhibited this increase in PGI(2) production. In conclusion, PCS gene transfer modulated COX-2-mediated prostanoid synthesis and inhibited neointimal formation after balloon injury.

DOI： 10.1161/hq0202.104123

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FEDERATION AMER SOC EXP BIOL  

The effect of uni-axial cyclic mechanical stretch on the activation of the transcription factor nuclear factor kappaB (NF-kappaB) was investigated in a human fibroblast cell line (TIG-1). In response to uni-axial cyclic stretch, NF-kappaB was found to be translocated into the nucleus. The NF-kappaB was first detectable 2 min after the onset of stretch and then peaked at 4 min and returned to the basal level within 10 min. To investigate whether NF-kappaB is activated following the translocation into the nucleus, we measured the luciferase activity in the cells transfected with pNF-kappaB-luciferase. The activity of luciferase increased 4 min after the initiation of cyclic stretch, peaked at 15 min (6.4-fold increase), and decreased gradually. We examined the involvement of the stretch-activated (SA) channel in the stretch-induced NF-kappaB activation. The application of Gd3+, a blocker of the SA channel, or the removal of extracellular Ca2+ inhibited both the translocation into the nucleus and the activation of NF-kappaB, which suggests that NF-kappaB is activated by uni-axial cyclic stretch via SA channel activation in human lung fibroblasts.

DOI： 10.1096/fj.01-0354fje

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

1 Human umbilical vein endothelial cells cultured on a transparent silicone chamber were subjected to a short stretch pulse (ca. 1 s, 5-25% stretch) of their substrate and following increases in intracellular Ca2+ concentration ([Ca2+](i)) were measured by fluorescence intensity ratiometry using fura-2.
2 In response to mechanical stretch, the cells in HEPES buffered saline exhibited a Ca2+ transient in a dose dependent way. The response was completely dependent on external Ca2+ and inhibited by gadolinium (Gd3+), suggesting that it was mediated by the activation of a stretch activated cation channel (SACatC).
3 Interestingly, the stretch induced Ca2+ transient was significantly augmented in the presence of basic polypeptide, protamine. This augmented Ca2+ response was inhibited neither by Gd3+ nor by the deprivation of external Ca2+, indicating that the SACatC is not responsible for this phenomenon.
4 In contrast, this augmentation was inhibited by depletion of intracellular Ca2+ stores with thapsigargin or by the pretreatment with phospholipase inhibitors such as U73122 and manoalide.
5 These results suggest the presence of a metabotropic mechanoreceptor distinct from the SACatC in vascular endothelium. This augmented [Ca2+](i) increase may contribute to the vasodilating response induced by protamine during heparin neutralization in cardiac surgery.

DOI： 10.1038/sj.bjp.0704386

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

We investigated the role of tyrosine phosphorylation of FAK in the stretch-induced MAPKs (extracellular signal-regulated kinase (ERK), p38MAPK) activation in mutant FAK-transfected fibroblasts. In response to uniaxial cyclic stretch (1 Hz, 120% in length), the levels of tyrosine phosphorylation of the Tyr-397 and Tyr-925 of FAK in control cells increased and peaked at 5 min (2.75 +/- 0.51, n = 3), and 20 min (2.98 +/- 0.58, n = 3), respectively, and the activities of MAPKs increased and peaked at approximately 10 min. On the other hand, in the mutant FAK-transfected cells, the stretch-induced MAPKs activation was significantly inhibited. The stretch-induced activation of MAPKs was also significantly abolished by either treatment with Gd3+ or extracellular Ca2+ removal which may inhibit intracellular Ca2+ increase caused by the activation of cation selective (Ca2+-permeable) stretch activated (SACatC) channels. These results suggest that the stretch-induced tyrosine-phosphorylation of FAK via SACatC activation is critical for the stretch-induced MAPKs activation. (C) 2001 Academic Press.

DOI： 10.1006/bbrc.2001.5775

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Superoxide production in response to cyclic stretch (1 Hz, 20 % in length) was investigated in human umbilical vein endothelial cells (HUVECs). The basal production of superoxide without stretch increased gradually, while the production of superoxide with stretch increased significantly as compared to that without stretch and it became significant 80 min after the onset of cyclic stretch (P&lt;0.05, n=8-14). The superoxide production increased in a stretch-dependent manner and became significant when stretch was more than 10 % (p&lt;0.05, n= 11-16). To investigate the involvement of SA channel, we added Gd3+ or EGTA in the reaction solution and examined the stretch-induced superoxide production. In cells stretched in the presence of 20 muM Gd3+, the stretch-induced superoxide production was significantly inhibited (at 120 min, p&lt;0.05, n=8-18). The cyclic stretch-induced superoxide production was also significantly inhibited by the removal of extracellular Ca2+ with 5 mM EGTA (at 120 min, p&lt;0.05, n = 8-18). Neither the application of Gd3+ nor the removal of extracellular Ca2+ significantly changed the basal production of superoxide. These data suggest that the stretch-induced superoxide production increases in time- and stretch-dependent manner and that the stretch-induced superoxide production in HUVECs is regulated by Ca2+ influx through SA channels. (C) 2001 Elsevier Science Inc. All rights reserved.

DOI： 10.1016/S0024-3205(01)01256-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Alternative models of cell mechanics depict the living cell as a simple mechanical continuum, porous filament gel, tensed cortical membrane, or tensegrity network that maintains a stabilizing prestress through incorporation of discrete structural elements that bear compression. Real-time microscopic analysis of cells containing GFP-labeled microtubules and associated mitochondria revealed that living cells behave like discrete structures composed of an interconnected network of actin microfilaments and microtubules when mechanical stresses are applied to cell surface integrin receptors, Quantitation of cell tractional forces and cellular prestress by using traction force microscopy confirmed that microtubules bear compression and are responsible for a significant portion of the cytoskeletal prestress that determines cell shape stability under conditions in which myosin light chain phosphorylation and intracellular calcium remained unchanged. Quantitative measurements of both static and dynamic mechanical behaviors in cells also were consistent with specific a priori predictions of the tensegrity model. These findings suggest that tensegrity represents a unified model of cell mechanics that may help to explain how mechanical behaviors emerge through collective interactions among different cytoskeletal filaments and extracellular adhesions in living cells.

DOI： 10.1073/pnas.141199598

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MACMILLAN PUBLISHERS LTD  

DOI： 10.1038/35082637

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.41.S208_1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

When exposed to a uni-axial cyclic stretch, cultured human umbilical vein endothelial cells (HUVECs) align and elongate perpendicular to the stretch axis. Previous studies showed that forskolin inhibited stretch-induced orientation of endothelial cells, suggesting that adenosine3:5-cyclic monophosphate (cAMP) plays an important role in the shape change. However, we have recently shown that stretch-induced shape changes in cultured HUVECs are due to increased [Ca2+],. In the present study, we examined the possible role of cAMP in stretch-induced shape changes in cultured HUVECs, Application of uni-axial cyclic stretch induced a gradual rise in cAMP reaching a peak level at 60 min after the onset of stretch. The adenylate cyclase activator, forskolin, increased the basal level of cAMP but inhibited the rise in [Ca2+](i) resulting in no cell shape changes. In contrast, N 6,2-dibutyryladenosine3:5-cyclic monophosphate (dbcAMP) enhanced the stretch-induced increase in cAMP and [Ca2+] and resulted in cell shape changes. On the other hand, 2'5'-dideoxyadenosine (DDA), an adenylate cyclase inhibitor, inhibited stretch-induced increases in cAMP and [Ca2+]i resulting in no cell shape changes. In summary, our data showed that cell shape changes were consistently dependent on [Ca2+](i) rather than cAMP levels. We conclude that the primary second messenger in the stretch-induced shape changes in HUVECs is intracellular Ca2+ rather than cAMP. (C) 2000 Elsevier Science Inc. All rights reserved.

DOI： 10.1016/S0024-3205(00)00844-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD  

The pathogenesis of pericyte loss, an initial deficit in the early stage of diabetic retinopathy, remains unclear. Polyol pathway hyperactivity has been implicated in the pathogenesis of diabetic retinopathy, and recent studies have suggested that apoptosis may be involved in pericyte loss. The present study was conducted to investigate whether high glucose induces apoptosis in cultured bovine retinal pericytes. The effect of an aldose reductase inhibitor. SNK-860, was also examined. After a 5 day incubation with various concentrations of glucose (5.5-40 mM) in the presence or absence of SNK-860, the cell viability and the percentages of dead cells were measured. and staining with the TUNEL method and Hoechst 33342, and DNA electrophoresis were performed. High glucose reduced the viability and increased the percentages of dead cells. TUNEL-positive cells were observed in pericytes under high glucose, but not in those under 5.5 mM glucose. In the staining of nuclei with Hoechst 33342, the percentage of apoptotic cells in total cells counted under high glucose was higher than that under 5.5 mM glucose. DNA electrophoresis of pericytes cultured with high glucose demonstrated a 'ladder pattern'. Hyperosmolarity also induced apoptosis in pericytes, but less than that by high glucose. SNK-860 inhibited the glucose-induced apoptosis in pericytes. These observations suggest that the pericyte loss in diabetic retinopathy involves an apoptotic process, and that the polyol pathway hyperactivity plays an important role in inducing apoptosis in pericytes by high glucose. (C) 2000 Academic Press.

DOI： 10.1006/exer.2000.0882

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1126/science.288.5470.1347

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC  

To examine the role of platelet-derived growth factor (PDGF) and the polyol pathway in the growth activity of smooth muscle cells (SMCs), [H-3]-thymidine incorporation, [I-125]-PDGF-BB binding and expression of PDGF-beta receptor protein were measured in rat aortic SMCs cultured with 5.5 or 20 mM glucose with or without anti-PDGF antibody or an aldose reductase inhibitor, epalrestat. SMCs cultured with 20 mM glucose demonstrated an accelerated thymidine incorporation compared with SMCs cultured with 5.5 mM glucose, which was prevented by anti-PDGF antibody. This acceleration of growth activity by 20 mM: glucose was accompanied by an increase in PDGF-BB binding, which was due to the increased number of PDGF-beta receptors and the overexpression of PDGF-beta receptor protein. Epalrestat prevented all these abnormalities. These observations suggest that polyol pathway hyperactivity plays an important role in the proliferation of SMCs which may be mediated through the accelerated expression of PDGF-beta receptor protein. (C) 1999 Academic Press.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC ADVANCEMENT SCIENCE  

Calcium-permeable, stretch-activated nonselective cation (SA Cat) channels mediate cellular responses to mechanical stimuli. However, genes encoding such channels have not been identified in eukaryotes. The yeast MID1 gene product (Mid1) is required for calcium influx in the yeast Saccharomyces cerevisiae. Functional expression of Midi in Chinese hamster ovary cells conferred sensitivity to mechanical stress that resulted in increases in both calcium conductance and the concentration of cytosolic free calcium. These increases were dependent on the presence of extracellular calcium and were reduced by gadolinium, a blocker of SA Cat channels. Single-channel analyses with cell-attached patches revealed that Midi acts as a calcium-permeable, cation-selective stretch-activated channel with a conductance of 32 picosiemens at 150 millimolar cesium chloride in the pipette. Thus, Midi appears to be a eukaryotic, SA Cat channel.

DOI： 10.1126/science.285.5429.882

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER PHYSIOLOGICAL SOC  

With the use of the patch-clamp technique, five kinds of stretch-activated (SA) ion channels were identified on the basis of their single-channel conductances and ion selectivities in cultured chick ventricular myocytes. Because a high-conductance K+-selective channel predominated among these channels, we concentrated on characterizing its properties mostly using excised inside-out patches. With 145 mM KCl solution in the pipette and the bath, the channel had a conductance of 199.8 +/- 8.2 pS (n = 22). The ion selectivities among K+, Na+, Ca2+, and Cl- as estimated from their permeability ratios were P-Na/P-K = 0.03, P-Ca/P-K = 0.025, and P-Cl/P-K = 0.026. The probability of the channel being open (P-0) increased with the Ca2+ concentration in the bath ([Ca2+](b); dissociation constant Kd = 0.51 mu M at +30 mV) and membrane potential (voltage at half-maximal P-o, = 39.4 mV at 0.35 FIM [Ca2+](b)). The channel was blocked by gadolinium, tetraethylammonium, and charybdotoxin from the extracellular surface and, consequently, was identified as a Ca2+-activated K+ (K-Ca) channel type. The channel was also reversibly activated by ATP applied to the intracellular surface (K-d = 0.74 mM at 0.10 mu M [Ca2+](b) at + 30 mV). From these data taken together, we concluded that the channel is a new type of K-Ca channel that could be designated as an "SA K-Ca,K-ATP channel. "To our knowledge, this is the first report of K-Ca channel in heart cells.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

When subjected to uni-axial cyclic stretch (120% in length, 1 Hz), fibroblasts (3Y1) aligned perpendicular to the stretch axis in a couple of hours. Concomitantly with this orienting response, protein tyrosine phosphorylation of cellular proteins (molecular masses of approximately 70 kDa and 120-130 kDa) increased and peaked at 30 minutes, Immuno-precipitation experiments revealed that paxillin, pp125(FAK), and pp130(CAS) were included in the 70 kDa, and 120-130 kDa bands, respectively. Treatment of the cells with herbimycin A, a tyrosine kinase inhibitor, suppressed the stretch induced tyrosine phosphorylation and the orienting response suggesting that certain tyrosine kinases are activated by stretch. We focused on pp60(src), the most abundant tyrosine kinase in fibroblasts. The kinase activity of pp60(src) increased and peaked at 20 minutes after the onset of cyclic stretch. Treatment of the cells with an antisense S-oligodeoxynucleotide (S-ODN) against pp60(src), but not the sense S-ODN, inhibited the stretch induced tyrosine phosphorylation and the orienting response. To further confirm the involvement of pp60(src), we performed the same sets of experiments using c-src-transformed 3Y1 (c-src-3Y1) fibroblasts. Cyclic stretch induced a similar orienting response in c-src-3Y1 to that in wild-type 3Y1, but with a significantly faster rate. The time course of the stretch-induced tyrosine phosphorylation was also much faster in c-src-3Y1. than in 3Y1 fibroblasts, These results strongly suggest that cyclic stretch induces the activation of pp60(src) and that pp60(src) is indispensable for the tyrosine phosphorylation of pp130(CAS), pp125(FAK) and paxillin followed by the orienting response in 3Y1 fibroblasts.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC  

To deliver nonnuclear proteins into the nucleus, we have examined the locations and number of nuclear localizing signals by use of simian virus 40 large T-antigen (SV40Ta) and yeast enhanced green fluorescent protein (yEGFP) in Saccharomyces cerevisiae as a model system. When only one SV40Ta was added to either the N- or C-terminus of yEGFP, the fluorescence of yEGFP was detected in both the nucleus and the cytoplasm. When two SV40Ta signals were added, one to the N-terminus and one to the C-terminus of yEGFP (SV40Ta-yEGFP-SV40Ta), the fluorescence of yEGFP was localized in only the nucleus. When the presequence of cytochrome oxidase subunit IV (pCOXIV) was inserted between the SV40Ta and the N-terminus of yEGFP (SV40Ta-pCOXIV-yEGFP-SV40Ta) in this construct, the fluorescence was located in both the nucleus and the cytoplasm, suggesting that the increased distance between the two SV40Ta signals decreased the efficiency of transport into the nucleus. When an additional SV40Ta signal was inserted between pCOXIV and yEGFP (SV40Ta-pCOXIV-SV49Ta-yEGFP), the fluorescence was localized only in the nucleus, indicating that two SV40Ta signals spaced by pCOXIV of 28 amino acid residues forming an alpha-helix are potent in transporting yEGFP into the nucleus. These results indicate that two SV40Ta signals spaced appropriately are essential for the efficient transport of the nonnuclear protein into the nucleus. (C) 1999 Academic Press.

DOI： 10.1006/bbrc.1999.0269

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

Prostacyclin (PGI(2)), a metabolite of arachidonic acid, has the vasoprotective effects of vasodilation, anti-platelet aggregation, and inhibition of smooth muscle cell proliferation. We hypothesized that an overexpression of endogenous PGI(2) may accelerate the recovery from endothelial damage and inhibit neointimal formation in the injured artery. To test this hypothesis, we investigated in vivo transfer of the PGI(2) synthase (PCS) gene into balloon-injured rat carotid arteries by a nonviral lipotransfection method. Seven days after transfection, a significant regeneration of endothelium was observed in the arteries transfected with a plasmid carrying the rat PCS gene (pCMV-PCS), but little regeneration was seen in those with the control plasmid carrying the lacZ gene (pCMV-lacZ) (percent luminal circumference lined by newly regenerated endothelium: 87.1+/-6.9% in pCMV-PCS-transfected vessels and 6.9+/-0.2% in pCMV-lacZ vessels, P&lt;0.001). BrdU staining of arterial segments demonstrated a significantly lower incorporation in pCMV-PCS-transfected vessels (7.5+/-0.3% positive nuclei in vessel cells) than in pCMV-lacZ (50.7+/-9.6%, P&lt;0.01). Moreover, 2 weeks after transfection, the PCS gene transfer resulted in a significant inhibition of neointimal formation (88% reduction in ratio of intima/media areas), whereas medial area was similar among the groups. Arterial segments transfected with pCMV-PCS produced significantly higher levels of 6-keto-PGF(1 alpha), the main metabolite of PGI(2), compared with the segments transfected with pCMV-lacZ (10.2+/-0.55 and 2.1+/-0.32 ng/mg tissue for pCMV-PCS and pCMV-placZ, P&lt;0.001). In conclusion, this study demonstrated that an in vivo PCS gene transfer increased the production of PGI(2) and markedly inhibited neointimal formation with accelerated reendothelialization in rat carotid arteries after balloon injury.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

Mechanical stress has been implicated as one of the growth regulators in the heart. We investigated the effect of cyclic stretch stimulation on morphology and orientation of cultured cardiocytes. Embryonic rat (17 days postcoital) cardiomyocytes cultured on silicone dishes were cyclically stretched to 120% in length at a frequency of 30 cycles/min. After 12 h, in the initial stage of cultivation, cardiocytes and intracellular myofibrils oriented parallel to the stretch direction. When the stretch stimulus was prolonged to 24-48 h, myofibrils that oriented perpendicular to the stretch direction emerged. Furthermore. when the cells were stretched only in the later stage (after 24 h of cultivation). both cells and myofibrils tended to orient perpendicular to the stretch direction. Next we examined the effects of chemical compounds on these phase related changes in myofibril orientation None of the drugs tested (H-7, HA-1004, staurosporine, herbimycin A, genistein, GdCl3, and EGTA) blocked the parallel orientation of myofibrils induced by the initial-stage stretch. By contrast, H-7, staurosporine, herbimycin A, and genistein did inhibit almost completely the perpendicular orientation of the myofibrils induced by the late-stage stretch, but HA-1004, GdCl3, or EGTA did not, Immunoblotting study using anti-phsophotyrosine antibody indicated that tyrosine phosphorylation of a protein of about 125 kDa was enhanced in a time-dependent manner by the late-stage stretch, but not by the initial-stage stretch. In conclusion: the alignment change induced by cyclic stretch depends on the stage of cultivation: with stretch in the initial stage (within 12 h), cells and myofibrils orient parallel to the stretch; with stretch in the later stage (after 24 h), they orient perpendicular to the stretch. The effect of stretch in the later stage is likely mediated by protein kinase C and tyrosine kinase pathways. (C) 1999 Academic Press.

DOI： 10.1006/jmcc.1998.0865

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

The kinase activity of c-src increased and peaked at 15 min after an application of uni-axial cyclic stretch in HUVECs followed by a translocation of c-src to Triton-insoluble fraction. Suppression of c-src by an antisense S-oligodeoxynucleotide inhibited the stretch-induced tyrosine phosphorylation and morphological changes. The stretch-induced increase in c-src activity was inhibited by FK506, a specific inhibitor for calcineurin, by Gd3+, a blocker for stretch activated channels, and by the extracellular Ca2+ depletion suggesting the involvement of SA channels. These results strongly suggest c-src plays an important role in the downstream of SA channel activation followed by the morphological changes. (C) 1998 Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(98)01528-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS AUST  

When cybrids with a point mutation, which locates in the tRNA(Leu(UUR)) gene of mtDNA and causes a mitochondrial encephalomyopathy (MELAS syndrome), were exposed to a high concentration of oxygen (95%), the peroxide production markedly increased by 6 h of oxygen exposure, whereas the peroxide production was similar among the cybrids under a normal concentration of oxygen. The peroxide production by oxygen exposure was enhanced particularly in cybrids with high proportions of the mutant mtDNA and low respiratory capacities. The appearance of apoptotic cells by oxygen exposure was high in cybrids with the impaired respiratory function due to the mutation. An antioxidant NAC successfully suppressed both the peroxide production and apoptosis. These results imply that the peroxide production plays an important role in inducing apoptosis in :ells carrying; the mtDNA mutation causing encephalomyopathy.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

In this study, critical signaling pathway required for the stretch induced morphological changes of human umbilical endothelial cells (HUVECs) was investigated. Uniaxial cyclic stretch (1 Hz, 20% in length) of the cells cultured on an elastic silicon membrane induced a gradual morphological change in the cells from a polygonal shape to an elongated spindle-like shape whose long axis was aligned perpendicular to the stretch axis, We found that protein tyrosine phosphorylation of cellular proteins increased and peaked at 20 min in response to cyclic stretch. Either treatment of cells with gadolinium (Gd3+), a potent blocker for stretch-activated channels, or removal of extracellular Ca2+ blocked the tyrosine phosphorylation of the proteins, suggesting that stretch-activated (SA) ion channels regulated stretch specific tyrosine phosphorylation, The major phosphorylated proteins had molecular masses of approximately 120-135 kDa, and 70 kDa, Immunoprecipitation experiments revealed that paxillin, focal adhesion kinase (pp125(FAK)) and pp130(CAS) were included in the 70 kDa and 120-135 kDa bands, respectively, The morphological change was inhibited by herbimycin A and genistein, inhibitors of tyrosine kinases, suggesting that tyrosine phosphorylation was required for the morphological change, In addition, the kinase activation of pp125(FAK) was observed in response to cyclic stretch, Moreover, suppression of pp125(FAK) expression by the antisense phosphorothioate oligodeoxynucleotides (S-ODN) in HUVECs resulted in inhibition of tyrosine phosphorylation of paxillin and the stretch-dependent morphological changes. These results suggest that an activation of tyrosine kinase(s) by an increase in intracellular Ca2+ and pp125(FAK) play a critical role in the unique morphological change specifically observed in endothelial cells subjected to uni-axial cyclic stretch.

DOI： 10.1038/sj.onc.1201950

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The present work was designed to elucidate the involvement of Ca2+permeable stretch-activated (SA) channels in the orienting response of endothelial cells to uniaxial cyclic stretch. Endothelial cells from human umbilical vein were cultured on an elastic silicone membrane and subjected to uniaxial cyclic stretch (120% in length, 1 Hz). The cells started to change their morphology 15 min after the onset of stretch, and &gt
90% of the cells oriented perpendicularly to the stretch axis after 2 h. Associated with the orienting response, cell elongation proceeded with a slower rate. Both of the orientating and elongating responses were largely inhibited by the removal of external Ca2+ or by Gd3+, a potent blocker for the SA channel, but not by nifedipine. Intracellular Ca2+ concentration ([Ca2+](i)) transiently increased in response to uniaxial stretch, and the basal [Ca2+](i) gradually increased during cyclic stretch. This Ca2+ response was inhibited by the removal of extracellular Ca2+ or by the addition of Gd3+. These results suggest that stretch-dependent Ca2+ influx through SA channels is essential in the stretch-dependent cell orientation and elongation.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER PHYSIOLOGICAL SOC  

The present work was designed to elucidate the involvement of Ca2+-permeable stretch-activated (SA) channels in the orienting response of endothelial cells to uniaxial cyclic stretch. Endothelial cells from human umbilical vein were cultured on an elastic silicons membrane and subjected to uniaxial cyclic stretch (120% in length, 1 Hz). The cells started to change their morphology 15 min after the onset of stretch, and &gt;90% of the cells oriented perpendicularly to the stretch axis after 2 h. Associated with the orienting response, cell elongation proceeded with a slower rate. Both of the orientating and elongating responses were largely inhibited by the removal of external Ca2+ or by Gd3+, a potent blocker for the SA channel, but not by nifedipine. Intracellular Ca2+ concentration ([Ca2+](i)) transiently increased in response to uniaxial stretch, and the basal [Ca2+](i) gradually increased during cyclic stretch. This Ca2+ response was inhibited by the removal of extracellular Ca2+ or by the addition of Gd3+. These results suggest that stretch-dependent Ca2+ influx through SA channels is essential in the stretch-dependent cell orientation and elongation.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The effect of uni-axial cyclic mechanical stretch on the expression of cyclooxygenases (COX) was investigated in a human lung fibroblast cell line (TIG-1). In response to uni-axial cyclic stretch, the level of COX2 mRNA significantly increased and peaked at 3 h (9.09 +/- 3.82-fold, mean +/- standard error, n = 6, compared with that at 1 h). The level of the expression of COX2 protein peaked at 6 h, whereas the level of COX1 protein was not significantly changed. The involvement of stretch-activated (SA) channel was investigated in the stretch-induced COX2 production. The application of Gd3+, a blocker for SA channel, or the removal of extracellular Ca2+ inhibited the production of COX2 mRNA without any effect on the production of COX1 or GAPDH mRNA. These data strongly suggest that COX2 expression is up-regulated by uni-axial cyclic stretch via the activation of SA channel in human lung fibroblasts. (C) 1998 Academic Press.

DOI： 10.1006/bbrc.1998.8335

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC CANCER RESEARCH  

Cell adhesion to the extracellular matrix appears to trigger a cascade of intracellular signalings. We have shown previously that treatment of ovarian cancer cells with peritoneal conditioned medium or purified fibronectin (FN) activated matrix metalloproteinase 9 secretion and, thereby, cancer cell invasion, By use of antisense oligonucleotides to focal adhesion kinase (FAK) and a dominant-negative mutant of ras (S17Nras), we found that both FAK and c-Ras were required for the activation of matrix metalloproteinase 9 secretion by FN. In addition, both antisense oligonucleotides to FAR and S17Nras inhibited mitogen-activated protein kinase activation hy FN treatment, suggesting the involvement of mitogen-activated protein kinase in the FN-dependent signaling.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER LUNG ASSOC  

Tumor cells expressing the herpes simplex virus-thymidine kinase (HSV-tk) gene become sensitive to ganciclovir (GCV), and the phenomenon by which tumor cells surrounding the HSV-tk expressing cells also become sensitive to CCV is known as the "bystander effect." The purpose of this study was to investigate the bystander effect in human lung-cancer cell lines, and the role of gap-junctional intercellular communication as the mechanism responsible for it. Gap-junctional intercellular communication was measured both with a dye-transfer assay involving single-cell microinjection of Lucifer Yellow and with a PKH26/calcein-AM double-dye-transfer assay. Significant bystander tumoricidal effect was observed in lung-cancer cell lines when cultured cells contained only 10% HSV-tk expressing cells. This was also observed to occur with cell lines of different origin or from different species. Although gap-junctional intercellular communication characterized by rapid transfer of Lucifer Yellow was not observed, we did detect gap-junctional communication marked by the slow transfer of calcein-AM in lung-cancer cell lines. However, neither an inhibitor (I-octanol) nor an enhancer (all trans-retinoic acid [ATRA]) of gap-junctional communication affected the extent of the bystander effect. These findings suggest that low levels of gap-junctional communication may be efficient for producing the bystander effect in lung-cancer cells, or chat other mechanisms may underlie this effect. Although gap-junctional communication may play an important role in generating the bystander effect in tumor cells expressing the HSV-tk gene, further knowledge of the mechanism of this effect may help improve the treatment of lung cancer with an HSV-tk system.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

The effect of uni-axial cyclic mechanical stretch on the expression of the adhesion protein integrin was investigated. Human umbilical endothelial cells (HUVECs) cultured an fibronectin coated silicon membranes were subjected to uni-axial cyclic stretch, The level of expression of integrin beta(3) mRNA was found to be increased and peaked at 4 hours in response to cyclic stretch using a semiquantitative RT-PCR method. The increased level of the integrin mRNA from stretched HUVECs remained higher than that ii om non-stretched controls. The amount of integrin beta(3) also increased and peaked at 12 hr, Immuno-fluorescent microscopy revealed that the amount of integrin beta(3) adhesions increased in stretched HUVECs compared with that in non-stretched HUVECs. These results suggest that uni-axial cyclic stretch up-regulates the expression of integrin beta(3). This increase in integrin beta(3), may enhance the adhesiveness to the substratum and contribute to the protection of HUVECs against being peeled off from the vessel wall. (C) 1997 Academic Press.

DOI： 10.1006/bbrc.1997.7364

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

We investigated the signaling mechanism of stretch-induced cell remodeling in human umbilical vein endothelial cells (HUVECs). Freshly dissociated HUVECs were cultured on an elastic silicon membrane and subjected to uniaxial cyclic stretch (20% in length, 1Hz). The cells started to change their morphology as early as 15 min after stretch onset, and most cells eventually aligned perpendicularly to the stretch axis within Ih. This remodeling was dependent on the increase in intracellular calcium concentration ([Ca2+](i)) via a Ca2+-permeable stretch-activated (SA) channel. During the process of remodeling, extensive rearrangement of stress fibers and focal adhesions was observed, which may be close to the final step in the intracellular signaling cascade. This event was [Ca2+](i)-dependent, suggesting the existence of a Ca2+-dependent intermediate cascade that links [Ca2+](i) to the rearrangement of cytoskeletons and focal adhesions. We found that some proteins, including pp125(FAK) (focal adhesion kinase) and paxillin, were tyrosine phosphorylated during cyclic stretch in a Ca2+-dependent manner. Inhibition of this tyrosine phosphorylation prohibited the stretch-dependent rearrangement of cytoskeletons and focal adhesions as well as the remodeling. Finally the tyrosine kinase src, which could phosphorylate pp125(FAK), was found to be activated in a [Ca2+](i)-dependent way during stretch. All of the above molecular events were consistently Ca2+-dependent, which led us to propose the signaling cascade: SA channel activation --&gt; [Ca2+](i) increase --&gt; src activation --&gt; protein tyrosine phosphorylation --&gt; rearrangement of cytoskeletons and focal adhesions --&gt; cell remodeling.

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Language：English   Publisher：Nagoya University  

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Language：English   Publisher：Nagoya University  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER PHYSIOLOGICAL SOC  

1. A characteristic plateau pattern was observed in the rate of afferent discharges during ramp-and-hold stretch of spindles isolated from semitendinosus muscles of frogs. The plateau pattern was more frequent in summer frogs (84%) than winter frogs (11%).
2. The plateau pattern consisted of a discharge rate around 120 imp/s at the end of dynamic stretch, followed by second and third steps of plateau rates around 60 and 40 imp/s, respectively. The intervals of impulses in lower steps were approximately n times those of the top step.
3. The plateau pattern was not sensitive to cutting extracapsular myelinated branches or lowering temperature from 23 to 12-degrees-C. However, the number of the plateau was reduced in both cases.
4. Application of depolarizing current to the sensory terminal abolished the plateau pattern. In contrast, in spindles that did not show a plateau pattern, hyperpolarizing current induced such a pattern.
5. Calcium channel blockers and protein kinase C inhibitors abolished the plateau pattern. The plateau pattern could be established in quiescent spindles by drugs eliciting Ca2+ entry, raising cytosolic-free Ca2+, and activating protein kinase C.
6. The most striking aspect of the present study is the stability in the discharge rate at each step of the plateau, irrespective of different experimental conditions. This suggests that the spindle sensory terminal possesses a stable intrinsic rhythm generator in excitation, of which maximum frequency is 120 imp/s. The generator seems to be triggered by stretch stimulus and to be regulated by cytoplasmic Ca2+ and protein kinase C.

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Endothelial cells are subjected to shear stresses by blood flow, normal stresses by blood pressure, and stretch by vessel expansion. These forces are known to induce secretions of several vasoactive substances probably via internal calcium mobilization (R. F. Furchgott. Circ. Res. 53: 557-573, 1983; M. J. Peach, A. L. Loeb, H. A. Singer, and J. Saye. Hypertension Dallas 7, Suppl. I: I-94-1-100, 1985.). Here we report that stretching cellular membranes increased intracellular Ca2+ concentration ([Ca2+]i) in human umbilical endothelial cells cultured on silicon membranes. Upon application of a stretch pulse (3-s duration), [Ca2+]i increased rapidly and decayed slowly. The following results suggest that this increase arises from Ca2+ entry through stretch-activated (SA) channels: 1) the Ca2+ response disappeared when extracellular Ca2+ was removed; 2) gadolinium (Gd3+), a blocker for cation-selective SA channels, blocked the response but nifedipine did not; and 3) externally applied Mn2+, which is known to permeate mechanosensitive channels but not Ca2+ channels, entered the intracellular space immediately after an application of mechanical stretch. The increase in [Ca2+]i was found to consist of at least two components: an initial fast component and a delayed slower component. Ryanodine inhibited the slow component. It is suggested that stretching the membrane primarily induced extracellular Ca2+ entry through SA channels followed by Ca2+ releases from intracellular Ca2+ stores.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CENTER ACADEMIC PUBL JAPAN  

Stretch activated (SA) channels are believed to be activated by tension in the membrane generated by membrane stretch. However, very few studies have been made on the quantitative estimation of the tension during membrane stretch. Here we present a method to evaluate the tension both in patch clamped and intact cell-membranes. The tension in patch clamped membranes was calculated from Laplace's law by knowing transmembrane pressure and the radius of patch-curvature. We also provide a simpler version for calculating the tension from the pressure and pipette radius. The tension in intact cell membranes was calculated from Hook's law based on the measurement of changes in cell surface area. The estimated tension required for activating SA channels in both types of membranes was found to be comparable suggesting that,the SA channel acts as a physiological mechanotransducer in intact cells.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER PHYSIOLOGICAL SOC  

The nonapeptide bradykinin (BK) excites a subset of dorsal root ganglion (DRG) neurons with putative nociceptive functions by stimulating an inward cation current. In addition, BK stimulates various intracellular signaling pathways including an elevation of intra-cellular Ca2+. In a DRG neuron x neuroblastoma hybrid cell (F-11), BK stimulates similar increases in intracellular [Ca2+] and inward current but also elicits a large transient outward current through Ca2+-activated K channels. We have investigated the mechanisms underlying differential expression of outward current responses in the two cell types at the single channel level. Although K(Ca) channel activity appears in inside-out patches from both cells exposed to Ca2+, BK applied to the extrapatch membrane of cell-attached patches activates K(Ca) channels in F-11 but not DRG neurons. Whereas single K(Ca) channels are quantitatively similar in terms of conductance, voltage-dependence, and sensitivity to tetraethylammonium, they differ in sensitivity to intracellular Ca2+. Channel activation in both cells requires at least four Ca2+ ions, but half-maximal activation occurs at slightly higher [Ca2+] for DRG neurons. The shift in the Ca2+ dose-response curve combined with the steep [Ca2+] dependence of channel open probability makes it less likely that a BK-induced rise in internal [Ca2+] induced will trigger a transient outward current and resultant hyperpolarization in a DRG neuron.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

This article presents a brief review on the electrophysiological analysis of the structural aspects of the voltage-dependent SR (sarcoplasmic reticulum) K+ channel. In the first half, early attempts to determine the physical dimensions of the ion conducting mechanism such as the mouth, narrow tunnel, or ion selective filter of the channel, are reviewed. The depicted cartoon of the SR K+ channel, as an extremely short, busy district with a big mouth on each side, is quite similar to the recently-obtained reconstructed structural image of the acetylcholine receptor channel. In the latter half, we introduce our recent attempts to draw a physical image of the gating mechanism of the SR K+ channel. We examined, for example, the location of the gate and the voltage sensor, and the relationship between them. It is suggested that the gate and the sensor are connected tightly and that the sensor would be exposed to the surface of the lumen side of SR when the gate opens. Finally, the issue of substates in SR K+ channel is discussed. It is implied that the substate-conductances reflect a partial occlusion of the pore by an intermediate-open gate.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

DOI： 10.1007/BF01868639

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/0921-8696(90)90005-N

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Responsible for pages：pp243-262   Language：English

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Responsible for pages：pp177-186   Language：Japanese

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DOI： 10.1540/jsmr.54.83

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Language：Japanese   Publishing type：Research paper, summary (national, other academic conference)   Publisher：Japanese Society for Medical and Biological Engineering  

<p>Recent advancement of mechanobiological research contributes to understanding and cure of diseases, leading to the birth of mechanomedicine. Here we introduce innovative mechano-medical technologies that pursue clinical use. In dermal regenerative medicine, we performed co-culture of dermal keratinocytes and fibroblasts with stretch stimulus, and found thickening of dermal layer and facilitated basal membrane formation. In cardiac regenerative medicine, we found that exosomes released from stem cells in response to mechanical stimulus excrete paracrine factors and micro RNAs, and thus enhance therapeutic effect of cell therapy in vivo. In reproductive medicine, we applied mechanical stimulus to fertilized eggs using a tilting embryo culture system, and confirmed enhancement of blastocyst development rate. Exhaustive gene analysis suggested that the enhancement is due to alteration of gene expression at epiblast. We continue to reveal the mechanisms of biological response to mechanical stimulus, and pursue clinical application of mechano-medical therapy.</p>

DOI： 10.11239/jsmbe.55Annual.340

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：American Institute of Mathematical Sciences  

Cell surface receptors are involved in numerous important biological processes including embryogenesis, tissue differentiation, and cellular homeostasis. Among them, mechanosensitive ion channels play an essential role in cellular functions of every cell including neurons, cardiomyocytes, and osteocytes. Here, we discuss types, roles, structures, and biophysical factors that affect the functions of mechanosensitive ion channels.

DOI： 10.3934/biophy.2016.1.63

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Language：Japanese   Publisher：The Japan Society of Mechanical Engineers  

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DOI： 10.11392/jsao.43.206

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：CELL PRESS  

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Language：Japanese   Publisher：日本生物工学会  

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Other Link： http://dl.ndl.go.jp/info:ndljp/pid/10518796

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：Japanese   Publisher：The Japan Society of Mechanical Engineers  

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Language：Japanese   Publisher：(公社)日本矯正歯科学会  

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Language：Japanese   Publisher：The Japan Society of Mechanical Engineers  

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Language：Japanese   Publisher：Japanese Society of Mammalian Ova Research  

Mammalian embryo culture is influenced by autocrine and paracrine effects and the diffusion of harmful byproducts. The physiological development of the embryo occurs in the Fallopian tube under dynamic conditions. To obtain high quality embryos in in vitro culture, dynamic culture systems (DCSs) have been developed which mimic the dynamic physiological conditions. In this review, embryo culture using DCSs in vitro and their culture results are discussed. From the culture results of DCSs that move the medium and embryos, we deduced that an increase in the diffusion rate is one of the reasons for an improvement in embryo development. Diffusion around the embryos could be investigated by monitoring pH changes in the medium during embryo culture. Application of moderate mechanical stimuli to embryos using DCSs would induce improvement in embryo development. To understand mechanosensing of mammalian embryos, mechanical stimuli and intracellular calcium concentrations in the embryos should be quantitatively evaluated. Finally, strategies to apply these insights and technologies to human embryo development are briefly discussed.

DOI： 10.1274/jmor.28.174

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER THORACIC SOC  

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Language：Japanese   Publisher：The Japan Society of Mechanical Engineers  

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Language：Japanese   Publisher：The Japan Society of Mechanical Engineers  

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Language：Japanese   Publisher：The Japan Society of Mechanical Engineers  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：CELL PRESS  

DOI： 10.1016/j.bpj.2009.12.2986

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：CELL PRESS  

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Language：Japanese   Publisher：(一社)日本生殖医学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：Japanese   Publisher：The Japan Society of Mechanical Engineers  

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Language：Japanese   Publisher：The Japan Society of Mechanical Engineers  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：SPRINGER TOKYO  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：SPRINGER TOKYO  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：SPRINGER TOKYO  

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Language：Japanese   Publisher：The Japan Society of Mechanical Engineers  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：Japanese   Publisher：社団法人日本生体医工学会  

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Language：English   Publisher：Japanese Circulation Society  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：BIOPHYSICAL SOCIETY  

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DOI： 10.1380/jsssj.28.204

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：Japanese   Publisher：The Institute of Electronics, Information and Communication Engineers  

We developed a pH/CO_2 sensor system using two ion sensitive field effect transistors (ISFETs) to evaluate the metabolic activity of cultured cells. ISFETs were placed in a flow-through chamber with a window to be attached to a glass-plate on which cells were cultured. Measurements were performed with a baseline solution of low buffer capacity. In the quantitative estimation of cellular products, we assumed that cells secreted CO_2, lactic acid, and sodium bicarbonate (NaHCO_3), and then lactic acid and NaHCO_3 proceeded to the neutralization reaction to form sodium lactate and additional CO_2. We applied this system to evaluate the metabolic activity of bovine aortic endothelial cells and rabbit aortic smooth muscle cells.

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Other Link： http://search.jamas.or.jp/link/ui/2007008183

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DOI： 10.4044/joma1947.118.1_23

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：BIOPHYSICAL SOCIETY  

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