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Evaluation of coculture model composed of sinusoidal endothelial cells and small hepatocytes
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Journal of Biomechanics 2006, Vol. 39 (Suppl 1)
6377 Mo-Tu, no. 28 (P63) Evaluation of coculture model composed of sinusoidal endothelial cells and small hepatocytes H. Asamia, R. Sudoa, T. Mitakab, M. Ikedaa, K. Tanishitaa. 1Dept. efSystem
Design Engineering Keio University, Kanagawa, Japan, 2Department of Pathophysiology, Cancer Research Institute, Sapporo Medical University School of Medicine, Sapporo, Japan Interaction of sinusoidal endothelial cells (SECs) and hepatocytes is fundamentally important for vascularization of tissue-engineered liver. In vivo, layers of hepatocytes and SECs are interacted to form a continuous threedimensional tissue structure. SECs have fenestrae that regulate the passage of serum molecules by their size. SECs are lacking diaphragm and basement membrane, which results in easy pass of the serum molecules. Therefore, both selective transport and high permeability are achieved spontaneously. In this study, we investigated the interaction of SECs and hepatocytes using coculture model to mimic hepatic tissues in vivo. SECs were cocultured with SHs that hepatic progenitor cells using cell culture inserts. Both cells are isolated from rats using the two-step collagenase perfusion method. Both sides of the inserts were coated with collagen. SHs were plated on one side of culture inserts. After culturing SHs for 2 weeks, SECs were plated on the other side. Immunofluorescent staining was carried out to identify SECs and SHs respectively after culturing for 2 days or 6 days. Confocal laser scanning microscopy three-dimensionally observed the stained cells. The results showed that SHs and SECs had a contact in membrane pores. This experimental model was useful to investigate cell morphogenesis of combined cells like liver tissue.
10.5. Mechanotransduction 7558 Mo-Tu, no. 29 (P63) Anisotropic response of cardiomyocytes to cyclic strain S. Senyo 1, B. Russell 1,2. 1Bioengineering Department, University of Illinois
at Chicago, Chicago, Illinois, USA, 2physiology & Biophysics Department, University of Illinois at Chicago, Illinois, USA Biomechanical forces play a dramatic role in cardiac muscle adaptation and disease. Relevant mechanical elements include tensile, compressive, and shear stress. The mechanism(s) by which cells detect complex cyclic mechanical strain remains poorly understood. Therefore the hypothesis is that the direction and rate of cyclic mechanical strain regulate protein phosphorylation and localization. The Flexcell stretch device administers cyclic strain of desired amplitude and frequency, which determines strain characteristics upon our cell culture model. We previously mimicked in vivo alignment of cardiac myocytes with microfabricated grooved architecture and here orient cultured neonatal rat myocytes to a uniaxial force vector either transverse or longitudinal to the cell polar axis. Focal adhesion kinase (FAK) is a membrane associating protein which has been shown to have a strain dependent increase in phosphorylation. Using western blot detection, we find strain rate dependence in cardiac myocytes as well with a 50% increase in FAK phosphorylation at tyrosine 397 site over 0.5 to 2.0 Hz (p < 0.05). Parallel studies show the magnitude of the response is less at high cell density. Our initial results show anisotropic effects with a 25% higher FAK phosphorylation due to strain across the transverse axis compared with the long axis (n =2). ELISA assay offering more sensitivity reports a similar trend in the phosphorylation level (n = 1) within 20 minutes. This work suggests that cardiac myocytes discriminate between both the rate of rise and direction of force. Funded by HL 077995, HL 64956, and HL 62426. 4966 Mo-Tu, no. 30 (P63) Plaque-prone hemodynamic induction of preproendothelin-1 gene is regulated at transcriptional and post-transcriptional levels R. da Silva, C. Chambaz, N. Stergiopulos, P. Silacci. Laboratory ef
Hemodynamics and Cardiovascular Technology, Swiss Federal Institute of Technology, Lausanne, Switzerland Various hemodynamic forces generated by a perturbed blood flow can trigger an endothelial dysfunction contributing to the focal development of atherosclerotic plaques. Shear stress is considered as one of the major hemodynamic forces acting on the endothelium. In vessel bifurcations, known to be plaqueprone areas, endothelial cells (ECs) are exposed to an oscillatory shear stress (OSS) characterized by a low shear stress value and a cyclic reversal flow. Previous studies from our laboratory and others have shown that OSS affects endothelial cell structure and function in vitro and in ex vive arteries by decreasing endothelial nitric oxide synthase (eNOS) gene expression, nitric-oxide-mediated vasorelaxation and by increasing the expression of the preproendothelin-1 (ppET-1) gene [1]. However, the underlying molecular mechanisms of OSS-induced ppET-1 gene expression have not yet been elucidated. In order to investigate the role of OSS effects on ppET-1 gene
Poster Presentations promoter and mRNA stability, bovine arterial endothelial cells (BAECs) were exposed to an OSS in a specialized perfusion system. Transfection analysis showed that a 156-bp proximal promoter contain all the necessary cis-acting elements conferring oscillatory shear stress sensitivity. Mutational analysis of this proximal promoter allowed the identification of a crucial role of an AP-1 binding site present in this fragment. In addition, oscillatory flow induced the appearance of an AP-l-binding complex, as revealed by electrophoretic mobility shift assay. Another important regulatory pathway determining the level of ppET-1 gene expression is post-transcriptional and involves the stabilization of ppET-1 mRNA through the 3'-UTR. In the presence of a RNA polymerase inhibitor (DRB), OSS significantly increases the steadystate ppET-1 mRNA levels as compared to static conditions. In conclusion, the present study demonstrated for the first time that plaqueprone hemodynamics induces ppET-1 gene expression by transcriptional and post-transcriptional regulatory mechanisms via activation of AP-1 and stabilization of mRNA. References [1] Gambillara V., Montorzi G., Haziza-Pigeon C., Stergiopulos N., Silacci E J Vasc IRes 2005; 42: 535-44. 5334 Mo-Tu, no. 31 (P63) A systems approach to bone mechanotransduction and osteoporosis K.C. Mynampati 1, P.V.S. Lee2. 1Graduate Programme in Bioengineering,
National University of Singapore, Singapore, 2Defence Medical & Environmental Research Institute, Singapore Physical forces regulate a plethora of physiological processes and deregulation of mechanical responses contributes to major diseases like osteoporosis. Osteoporosis is a multi-factorial, age-related metabolic bone disease characterized by a reduction in bone mass and abnormal bone remodeling. Mechanical loads generated by gravity and locomotion stimulate bone remodeling to maintain optimal mechanical performance. Reduced mechanical stimulation due to sedentary lifestyle, limb paralysis, or space flight results in net bone loss through reduced bone formation and increased bone resorption. Mechanical stimulus regulates various cellular physiological functions including gene induction, protein synthesis, proliferation, and differentiation. Hence understanding how mechanical stimuli affect biological functions, a process commonly known as mechanotransduction, might be the key to elucidate basic bone biology and to devise new treatments for degenerative diseases like osteoporosis and osteoarthritis. The focus of the current work is to investigate bone mechanotransduction at the systems level, in the context of osteoporosis. Systems biology tools are used to develop dynamic "systems-lever' computational models of the biological responses (signaling pathways) during mechanotransduction. We have generated a novel "systems-theoretic" computational model of signaling pathways in osteoblasts and osteoclasts, based on Michaelis-Menten enzyme kinetics in SIMULINK ®. Systems theory techniques are used to model the crosstalk and feedback among the pathways. Different case scenarios are generated to simulate abnormal bone remodeling conditions commonly observed in osteoporosis. The preliminary results are in concordance with the existing literature. Such "systems-lever' quantitative models provide the benefits of quantitative and predictive analysis to elucidate the role of mechanotransduction in the pathogenesis of osteoporosis. 6804 Mo-Tu, no. 32 (P63) Development of a bioreactor for the application of Hydrostatic Pressure and Stretch (HyPaS) on monolayer cell cultures J. Eyckmans 1, P. Spaepen2, H. Van Oosterwyck2, EP. Luyten 1. 1Laboratory
for Skeletal Development and Joint Disorders, K.U.Leuven, Leuven, Belgium, 2Division of Biomechanics and Engineering Design, K.U.Leuven, Leuven, Belgium Bioreactors have been developed to study mechanotransduction in cells, subjected to different loading modes such as hydrostatic pressure, compression, stretch or shear. Unfortunately most systems can only apply one loading mode. To investigate the ability of cells to distinguish between different mechanical stimuli, we have developed a bioreactor that can subject cells to hydrostatic pressure (HP), stretch or their combination (pressure induced stretch, PIS). The bioreactor is composed of an upper and a lower compartment which is separated by a FlexcellTM 6-well plate. In the upper compartment the air (with 5% CO2) can be pressurized. Two holes connect the upper and the lower compartment to allow HP when open or PIS when closed. Loading posts under the membrane allow uniaxial stretch when vacuum is applied in the lower compartment. To quantify the strain magnitude and direction under stretch, Flexcell TM plates were spray-painted. Subsequently vacuum was applied and pictures of the unloaded and loaded membrane were taken through an inverted microscope by a CCD camera (SPOT). Displacement vectors and Green-Lagrange tensors were calculated in MatLab. A vacuum of
Journal of Biomechanics 2006, Vol. 39 (Suppl 1)
6377 Mo-Tu, no. 28 (P63) Evaluation of coculture model composed of sinusoidal endothelial cells and small hepatocytes H. Asamia, R. Sudoa, T. Mitakab, M. Ikedaa, K. Tanishitaa. 1Dept. efSystem
Design Engineering Keio University, Kanagawa, Japan, 2Department of Pathophysiology, Cancer Research Institute, Sapporo Medical University School of Medicine, Sapporo, Japan Interaction of sinusoidal endothelial cells (SECs) and hepatocytes is fundamentally important for vascularization of tissue-engineered liver. In vivo, layers of hepatocytes and SECs are interacted to form a continuous threedimensional tissue structure. SECs have fenestrae that regulate the passage of serum molecules by their size. SECs are lacking diaphragm and basement membrane, which results in easy pass of the serum molecules. Therefore, both selective transport and high permeability are achieved spontaneously. In this study, we investigated the interaction of SECs and hepatocytes using coculture model to mimic hepatic tissues in vivo. SECs were cocultured with SHs that hepatic progenitor cells using cell culture inserts. Both cells are isolated from rats using the two-step collagenase perfusion method. Both sides of the inserts were coated with collagen. SHs were plated on one side of culture inserts. After culturing SHs for 2 weeks, SECs were plated on the other side. Immunofluorescent staining was carried out to identify SECs and SHs respectively after culturing for 2 days or 6 days. Confocal laser scanning microscopy three-dimensionally observed the stained cells. The results showed that SHs and SECs had a contact in membrane pores. This experimental model was useful to investigate cell morphogenesis of combined cells like liver tissue.
10.5. Mechanotransduction 7558 Mo-Tu, no. 29 (P63) Anisotropic response of cardiomyocytes to cyclic strain S. Senyo 1, B. Russell 1,2. 1Bioengineering Department, University of Illinois
at Chicago, Chicago, Illinois, USA, 2physiology & Biophysics Department, University of Illinois at Chicago, Illinois, USA Biomechanical forces play a dramatic role in cardiac muscle adaptation and disease. Relevant mechanical elements include tensile, compressive, and shear stress. The mechanism(s) by which cells detect complex cyclic mechanical strain remains poorly understood. Therefore the hypothesis is that the direction and rate of cyclic mechanical strain regulate protein phosphorylation and localization. The Flexcell stretch device administers cyclic strain of desired amplitude and frequency, which determines strain characteristics upon our cell culture model. We previously mimicked in vivo alignment of cardiac myocytes with microfabricated grooved architecture and here orient cultured neonatal rat myocytes to a uniaxial force vector either transverse or longitudinal to the cell polar axis. Focal adhesion kinase (FAK) is a membrane associating protein which has been shown to have a strain dependent increase in phosphorylation. Using western blot detection, we find strain rate dependence in cardiac myocytes as well with a 50% increase in FAK phosphorylation at tyrosine 397 site over 0.5 to 2.0 Hz (p < 0.05). Parallel studies show the magnitude of the response is less at high cell density. Our initial results show anisotropic effects with a 25% higher FAK phosphorylation due to strain across the transverse axis compared with the long axis (n =2). ELISA assay offering more sensitivity reports a similar trend in the phosphorylation level (n = 1) within 20 minutes. This work suggests that cardiac myocytes discriminate between both the rate of rise and direction of force. Funded by HL 077995, HL 64956, and HL 62426. 4966 Mo-Tu, no. 30 (P63) Plaque-prone hemodynamic induction of preproendothelin-1 gene is regulated at transcriptional and post-transcriptional levels R. da Silva, C. Chambaz, N. Stergiopulos, P. Silacci. Laboratory ef
Hemodynamics and Cardiovascular Technology, Swiss Federal Institute of Technology, Lausanne, Switzerland Various hemodynamic forces generated by a perturbed blood flow can trigger an endothelial dysfunction contributing to the focal development of atherosclerotic plaques. Shear stress is considered as one of the major hemodynamic forces acting on the endothelium. In vessel bifurcations, known to be plaqueprone areas, endothelial cells (ECs) are exposed to an oscillatory shear stress (OSS) characterized by a low shear stress value and a cyclic reversal flow. Previous studies from our laboratory and others have shown that OSS affects endothelial cell structure and function in vitro and in ex vive arteries by decreasing endothelial nitric oxide synthase (eNOS) gene expression, nitric-oxide-mediated vasorelaxation and by increasing the expression of the preproendothelin-1 (ppET-1) gene [1]. However, the underlying molecular mechanisms of OSS-induced ppET-1 gene expression have not yet been elucidated. In order to investigate the role of OSS effects on ppET-1 gene
Poster Presentations promoter and mRNA stability, bovine arterial endothelial cells (BAECs) were exposed to an OSS in a specialized perfusion system. Transfection analysis showed that a 156-bp proximal promoter contain all the necessary cis-acting elements conferring oscillatory shear stress sensitivity. Mutational analysis of this proximal promoter allowed the identification of a crucial role of an AP-1 binding site present in this fragment. In addition, oscillatory flow induced the appearance of an AP-l-binding complex, as revealed by electrophoretic mobility shift assay. Another important regulatory pathway determining the level of ppET-1 gene expression is post-transcriptional and involves the stabilization of ppET-1 mRNA through the 3'-UTR. In the presence of a RNA polymerase inhibitor (DRB), OSS significantly increases the steadystate ppET-1 mRNA levels as compared to static conditions. In conclusion, the present study demonstrated for the first time that plaqueprone hemodynamics induces ppET-1 gene expression by transcriptional and post-transcriptional regulatory mechanisms via activation of AP-1 and stabilization of mRNA. References [1] Gambillara V., Montorzi G., Haziza-Pigeon C., Stergiopulos N., Silacci E J Vasc IRes 2005; 42: 535-44. 5334 Mo-Tu, no. 31 (P63) A systems approach to bone mechanotransduction and osteoporosis K.C. Mynampati 1, P.V.S. Lee2. 1Graduate Programme in Bioengineering,
National University of Singapore, Singapore, 2Defence Medical & Environmental Research Institute, Singapore Physical forces regulate a plethora of physiological processes and deregulation of mechanical responses contributes to major diseases like osteoporosis. Osteoporosis is a multi-factorial, age-related metabolic bone disease characterized by a reduction in bone mass and abnormal bone remodeling. Mechanical loads generated by gravity and locomotion stimulate bone remodeling to maintain optimal mechanical performance. Reduced mechanical stimulation due to sedentary lifestyle, limb paralysis, or space flight results in net bone loss through reduced bone formation and increased bone resorption. Mechanical stimulus regulates various cellular physiological functions including gene induction, protein synthesis, proliferation, and differentiation. Hence understanding how mechanical stimuli affect biological functions, a process commonly known as mechanotransduction, might be the key to elucidate basic bone biology and to devise new treatments for degenerative diseases like osteoporosis and osteoarthritis. The focus of the current work is to investigate bone mechanotransduction at the systems level, in the context of osteoporosis. Systems biology tools are used to develop dynamic "systems-lever' computational models of the biological responses (signaling pathways) during mechanotransduction. We have generated a novel "systems-theoretic" computational model of signaling pathways in osteoblasts and osteoclasts, based on Michaelis-Menten enzyme kinetics in SIMULINK ®. Systems theory techniques are used to model the crosstalk and feedback among the pathways. Different case scenarios are generated to simulate abnormal bone remodeling conditions commonly observed in osteoporosis. The preliminary results are in concordance with the existing literature. Such "systems-lever' quantitative models provide the benefits of quantitative and predictive analysis to elucidate the role of mechanotransduction in the pathogenesis of osteoporosis. 6804 Mo-Tu, no. 32 (P63) Development of a bioreactor for the application of Hydrostatic Pressure and Stretch (HyPaS) on monolayer cell cultures J. Eyckmans 1, P. Spaepen2, H. Van Oosterwyck2, EP. Luyten 1. 1Laboratory
for Skeletal Development and Joint Disorders, K.U.Leuven, Leuven, Belgium, 2Division of Biomechanics and Engineering Design, K.U.Leuven, Leuven, Belgium Bioreactors have been developed to study mechanotransduction in cells, subjected to different loading modes such as hydrostatic pressure, compression, stretch or shear. Unfortunately most systems can only apply one loading mode. To investigate the ability of cells to distinguish between different mechanical stimuli, we have developed a bioreactor that can subject cells to hydrostatic pressure (HP), stretch or their combination (pressure induced stretch, PIS). The bioreactor is composed of an upper and a lower compartment which is separated by a FlexcellTM 6-well plate. In the upper compartment the air (with 5% CO2) can be pressurized. Two holes connect the upper and the lower compartment to allow HP when open or PIS when closed. Loading posts under the membrane allow uniaxial stretch when vacuum is applied in the lower compartment. To quantify the strain magnitude and direction under stretch, Flexcell TM plates were spray-painted. Subsequently vacuum was applied and pictures of the unloaded and loaded membrane were taken through an inverted microscope by a CCD camera (SPOT). Displacement vectors and Green-Lagrange tensors were calculated in MatLab. A vacuum of