Mounting evidence suggests that the normal biomechanical state of arteries may include a nearly equibiaxial intramural stress, and that arteries tend to undergo rapid and dramatic remodeling when perturbed from this normal state. Technical developments in the early 1980s and late 1990s enabled in vitro and ex vivo studies, respectively, of isolated perfused microvessels, and it is clear that they share many similarities in behavior with arteries. To date, however, there has been no systematic study of the effects of biaxial loading on the biomechanical behavior of arterioles. In this project, we describe a modification to a prior in vitro arteriole test system that allowed us to investigate the role of altered axial stretch on the passive, myogenic, and fully contracted biaxial behavior of isolated rat cremaster arterioles. We show that axial stretches from 85% to 110% of normal values induce modest changes in the measured circumferential and axial stress-stretch behavior and similarly in traditional measures of distensibility and myogenic index. Nevertheless, altered axial stretch has a dramatic affect on the biaxial state of stress and it appears that near equibiaxial stress occur at axial stretches larger than those used previously. Whereas this finding will not affect prior estimates of material and functional behavior, it may have important implications for the design of long-term ex vivo and in vivo studies wherein vessel growth and remodeling are critical.

To better study frequency related effects such as attenuation and tuning, we developed a frequency dependent seismic reflection analysis. Comprehensive tests on full waveform synthetics and observations from the Teal South ocean bottom seismic (OBS) data set confirmed that normal moveout (NMO) stretch could distort both frequency and amplitude information severely in shallow events and far offset traces. In synthetic tests, our algorithm recovered amplitude and frequency information ac-curately. This simple but robust target oriented NMO stretch correction scheme can be used on top of an existing seismic processing flow for further analyses. By combining the NMO stretch correction, spectral decomposition, and crossplots of am-plitude versus offset (AVO) attributes, we tested the frequency dependent workflow over Teal south and Ursa field data sets for improved reservoir characterization. As expected from NMO stretch characteristics, low frequencies have been less affected while mid and high frequency ranges were affected considerably. In seismic attribute analysis, the AVO crossplots from spectrally decomposed prestack data confirmed the improved accuracy and effectiveness of our workflow in mid and high frequency regions. To overcome poor spectral decomposition results due to low signal to noise ratio (S/N) in the Teal South application, we also implemented a substack scheme that stacks adjacent traces to increase S/N ratio while reducing the amount of data to process and increasing the accuracy of the spectral decomposition step. Synthetic tests verified the effectiveness of this additional step. An application to the Ursa, Gulf of Mexico, deep water data set showed significant improvement in high frequency data while correcting biased low frequency information.

Cells in the body respond to mechanical loads in ways that are crucial to normal and disease physiology. Understanding these processes is difficult due to the complex mechanical environment in vivo. In this research, we have developed several cell-stretching devices capable of subjecting cell cultures to non-uniform stretch distributions in order to investigate pathological responses of vascular smooth muscle cells to physiologic stretches. 10T1/2 cells were cyclically stretched with these devices for 24 hours upon silicone membranes, PDMS tubes, and within 3D PEGDA hydrogels. After stretching, altered cell behaviors were measured, including orientation, proliferation (quantified by BrdU incorporation), and gene expression (quantified by real-time, RT-PCR). Cells demonstrated marked changes in orientation, proliferation, and mRNA expression, which all varied with cellular location in the non-uniform environment. More specifically, increased orientation, increased proliferation, and more dramatically altered mRNA expression were found in regions of high, uniaxial stretch, relative to regions of low, near-equibiaxial stretch. These findings demonstrate the capabilities of graded stretch distributions to produce graded cell responses, indicating potentially localized smooth muscle cell behavior in a diseased artery. The novel devices employed herein will hopefully improve our understanding of these complicated cellular pathways, ultimately allowing for improved treatment or prevention of vascular disease.

The lymphatic system plays an important role in fluid and protein balance within the interstitial spaces. Its dysfunction could result in a number of debilitating diseases, namely lymphedema. Lymphatic vessels utilize both intrinsic and extrinsic mechanisms to pump lymph. Intrinsic pumping involves the active contraction of vessels, a phenomenon that is regulated in part by nitric oxide (NO) produced by lymphatic endothelial cells (LECs). NO production by arterial endothelial cells has been shown to be sensitive to both shear stress and stretch. Therefore, because of the unique mechanical environment of the LECs, we hypothesize that mechanical forces play an important role in regulation of the lymphatic pumping. Parallel-plate flow chambers and indenter-based cyclic stretch devices were constructed and used to apply mechanical loads to LECs. In addition, high-throughput micro-scale channels were developed and tested for shear experiments to address the need to increase the productivity and high- resolution imaging. Twenty-four hours treatment of LECs with different shear stress conditions showed a shear-dependent elevation in NO production. Moreover, 2.5 folds increase in cumulative NO was observed for stretched cells compared to the unstretched cells over six hours period. In conclusion, the upregulation observed in NO production under mechanical stimuli suggest new regulatory mechanisms that can be pharmaceutically targeted. These results provide an unprecedented insight into lymphatic pumping mechanism.

Age-related muscle atrophy and the importance of satellite cells in muscle maintenance, growth and repair led us to examine the effects of mechanical stretch, nitric oxide (NO), and age on satellite cell (SC) activation and gene expression in normal young and old mice. Baseline variables (body mass, muscle mass, fiber cross-sectional area (CSA), muscle strength, SC population, stretch activation and gene expression) were obtained from normal C57BL/6 mice at 3-, 8-, 12- and 18-months-of-age. Activation was assayed by 3H-thymidine incorporation into extensor digitorum longus (EDL) muscles isolated for culture. In a second experiment, muscle from 8- and 18-month-old mice was treated with one or more of: stretch; NO-donors (L-Arginine (LA), isosorbide dinitrate (ISDN)) and; Nω-nitro-L-Arginine methyl ester (LN). EDL muscles from 6-month-old mice required a greater stretch stimulus (20% vs. 10% length increase) than EDL from younger mice to increase SC activation. Stretch did not increase SC activation in mice older than 6 months-of-age. NO supplementation from an exogenous source (ISDN) increased SC activation by stretch in 8- but not 18-mo-old EDLs. In a third experiment, 8- and 18-month-old mice were subjected to 3 weeks of voluntary wheel running, or not. The EDL, tibialis anterior (TA), gastrocnemius (GAST) and quadriceps (QUAD) muscles were selected for analysis following sacrifice. The QUAD muscle from 8-month-old mice was the only muscle that demonstrated an exercise-induced increase in SC activation, elevated expression of neuronal nitric oxide synthase (NOS-I) and downregulation of myostatin, a gene that inhibits muscle growth. These results suggest mechanical stimulation of satellite cells and regulation of gene expression that controls muscle growth in voluntary contractile tissue is muscle-specific and age-dependent. / May 2009

Arterial endothelial cells (ECs) are subjected to pulsatile strain due to pressure changes in the cardiac cycle and this may play a significant role in vascular function in health and disease. Further, ECs differentially respond to different patterns of strain. There is much evidence that cyclic uniaxial strain results in a perpendicular orientation of ECs and their stress fibers, while no such alignment occurs in response to cyclic equaibiaxial stretch. It is unclear how cells and their stress fibers determine their specific response to particular spatiotemporal changes in the matrix, however. Given that ECs located at regions in the arterial tree prone to atherogenesis are non-aglined, while ECs in relatively healthy regions are oriented perpendicular to the principal direction of cyclic stretch, it is important to understand the mechanisms which regulate stretch-induced stress fiber alignment. The focus of this thesis was to develop realistic models to describe the dynamic changes in the organization of stress fibers in response to diverse spatiotemporal patterns of stretch. The model is based on the premise that stress fibers are pre-stressed at a ?homeostatic? level so that stress fibers are extended beyond their unloaded lengths, and that perturbation in stress fiber length from the homeostatic level destabilizes the stress fibers. A deterministic model described experimentally measured time courses of stress fiber reorientation perpendicular to the direction of cyclic uniaxial stretch, as well as the lack of alignment in response to equibiaxial stretch. In the case of cyclic simple elongation with transverse matrix contraction, stress fibers oriented in the direction of least perturbation in stretch. Model analysis indicated the need for a time-dependent stress fiber mechanical property, however. Thus, a stochastic model was developed that incorporated the concept that stress fibers tend to self-adjust to an equilibrium level of extension when they are perturbed from their unload lengths with the turnover of stress fibers. The stochastic model successfully described experimentally measured time courses of stress fiber reorganization over a range of frequencies. At a frequency of 1 Hz, stress fibers predominantly oriented perpendicular to stretch, while at 0.1 Hz the extent of stress fiber alignment was markedly reduced and at 0.01 Hz there was no alignment at all. Both the deterministic and stochastic models accurately described the relationship between stretch magnitude and the extent of stress fiber alignment in endothelial cells subjected to cyclic uniaxial stretch. Parameter sensitivity analyses for each model were used to demonstrate the effects of each parameter on the characteristics of the system response. In summary, the mathematical models were capable of describing stress fiber reorganization in response to diverse temporal and spatial patterns of stretch. These models provide a theoretical framework to elucidate the mechanisms by which adherent cells sense the characteristics of matrix deformation and describe a mechanism by which the cells can then adapt to such deformations to maintain mechanical homeostasis.

Mounting evidence suggests that the normal biomechanical state of arteries may include a nearly equibiaxial intramural stress, and that arteries tend to undergo rapid and dramatic remodeling when perturbed from this normal state. Technical developments in the early 1980s and late 1990s enabled in vitro and ex vivo studies, respectively, of isolated perfused microvessels, and it is clear that they share many similarities in behavior with arteries. To date, however, there has been no systematic study of the effects of biaxial loading on the biomechanical behavior of arterioles. In this project, we describe a modification to a prior in vitro arteriole test system that allowed us to investigate the role of altered axial stretch on the passive, myogenic, and fully contracted biaxial behavior of isolated rat cremaster arterioles. We show that axial stretches from 85% to 110% of normal values induce modest changes in the measured circumferential and axial stress-stretch behavior and similarly in traditional measures of distensibility and myogenic index. Nevertheless, altered axial stretch has a dramatic affect on the biaxial state of stress and it appears that near equibiaxial stress occur at axial stretches larger than those used previously. Whereas this finding will not affect prior estimates of material and functional behavior, it may have important implications for the design of long-term ex vivo and in vivo studies wherein vessel growth and remodeling are critical.

To better study frequency related effects such as attenuation and tuning, we developed a frequency dependent seismic reflection analysis. Comprehensive tests on full waveform synthetics and observations from the Teal South ocean bottom seismic (OBS) data set confirmed that normal moveout (NMO) stretch could distort both frequency and amplitude information severely in shallow events and far offset traces. In synthetic tests, our algorithm recovered amplitude and frequency information ac-curately. This simple but robust target oriented NMO stretch correction scheme can be used on top of an existing seismic processing flow for further analyses. By combining the NMO stretch correction, spectral decomposition, and crossplots of am-plitude versus offset (AVO) attributes, we tested the frequency dependent workflow over Teal south and Ursa field data sets for improved reservoir characterization. As expected from NMO stretch characteristics, low frequencies have been less affected while mid and high frequency ranges were affected considerably. In seismic attribute analysis, the AVO crossplots from spectrally decomposed prestack data confirmed the improved accuracy and effectiveness of our workflow in mid and high frequency regions. To overcome poor spectral decomposition results due to low signal to noise ratio (S/N) in the Teal South application, we also implemented a substack scheme that stacks adjacent traces to increase S/N ratio while reducing the amount of data to process and increasing the accuracy of the spectral decomposition step. Synthetic tests verified the effectiveness of this additional step. An application to the Ursa, Gulf of Mexico, deep water data set showed significant improvement in high frequency data while correcting biased low frequency information.

Arteries possess the ability to grow and remodel in response to sustained alterations in biomechanical loading, likely via mechanisms that are similarly involved in diverse arterial pathologies and responses to treatment. In particular, myriad experminental observations suggest that cell and matrix turnover within vasoaltered states enable arteries to adapt to sustained changes in mechanical stimuli. The goal herein is to show explicitly how altered smooth muscle contractility and matrix growth and remodeling work together to adapt the geometry, structure, stiffness, and function of a representative basilar artery. This work seeks to illustrate the importance of complementary vasoactivity and matrix remodeling for basilar arteries in response to sustained alterations in mechanical stimuli. Toward this end, an extended constrained mixture model of the arterial wall is employed whereby the mass fractions, material properties, and natural configurations of individual constituents can evolve separately and thereby dictate overall growth and remodeling. This approach accounts for fundamentally important behaviors. Simulations provide important intuition and insight regarding constitutive functional forms and model parameters.

"Uniaxial Cyclic Stretch-Stimulated Glucose Transport Is Mediated by a Ca2+-Dependent Mechanism in Cultured Skeletal Muscle Cells" Pathobiology, v.74, n.3, pp.159-168を、博士論文として提出したもの。 / 名古屋大学博士学位論文 学位の種類:博士（リハビリテーション療法学）（課程）学位授与年月日:平成19年3月23日