Takeo Matsumoto

Stress and Strain Distribution in Hypertensive and Normotensive Rat Aorta Considering Residual Strain

The effects of hypertension on the stress and strain distributions through the wall thickness were studied in the rat thoracic aorta. Goldblatt hypertension was induced by constricting the left renal artery for 8 weeks. Static pressure-diameter-axial force relations were determined on excised tubular segments. The segments were then sliced into thin ring specimens. Circumferential strain distributions were determined from the cross-sectional shape of the ring specimens observed before and after releasing residual stresses by radial cutting. Stress distributions were calculated using a logarithmic type of strain energy density function. The wall thickness at the systolic blood pressure, P(sys) significantly correlated with P(sys). The mean stress and strain developed by P(sys) in the circumferential direction were not significantly different between the hypertensive and control aortas, while those in the axial direction were significantly smaller in the hypertensive aorta than in the control. The opening angles of the stress free ring specimens correlated well with P(sys). The stress concentration factor in the circumferential direction was almost constant and independent of P(sys) although the stress distributions were not uniform through the wall thickness. Histological observation showed that the wall thickening caused by hypertension is mainly due to the hypertrophy of the lamellar units of the media, especially in the subintimal layer where the stress increase developed by hypertension is larger than in the other layers. These results indicate that: (a) the aortic wall adapts itself to the mechanical field by changing not only the wall dimensions but also the residual stresses, (b) this adaptation is primarily related to the circumferential stress but not to the axial stress, and (c) the aortic smooth muscle cells seem to change their morphology in response to the mechanical stress.

The pipette aspiration applied to the local stiffness measurement of soft tissues

A simple method of identifying the initial slope of the stress-strain curve (i.e., Youngs modulus of the soft tissue) by introducing the pipette aspiration technique is presented. The tissue was assumed to be isotropic and macroscopically homogeneous. Numerical simulations by the linear finite element analysis were performed for the axisymmetric model to survey the effects of friction at the tissue-pipette contact boundary, pipette cross-sectional geometry, relative size of the specimen to the pipette, and the layered inhomogeneity of the specimen tissue. The friction at the contact region had little effect on the measurement of Youngs modulus. The configuration of the pipette was shown to affect the measurement for small pipette wall thickness. The measurement also depended on the relative size of the specimen to the pipette for relatively small specimens. The extent of the region contributing to the measurement was roughly twice the inside radius of the pipette. In this region, the maximum stress did not exceed the level of the aspiration pressure, with only minor exceptional locations. Calculation of strain energy components indicated that the major contributions to the deformation under pipette aspiration were by the normal extension and shear deformation in pipette axial direction. Experimental verification of the present method for the isotropic, homogeneous artificial material is also presented.

Local elastic modulus of atherosclerotic lesions of rabbit thoracic aortas measured by pipette aspiration method

Changes in mechanical properties of arteries during atherogenesis remain controversial. One of the reasons could be that they have been evaluated with parameters measured in a whole vessel, although the lesions are localized. The local elastic modulus of atherosclerotic lesions was measured by the pipette aspiration method in thoracic aortas of rabbits fed a cholesterol diet for 8, 16, 24 and 28 weeks. The global elastic modulus of the whole aorta was measured by the pressure-diameter test. The local modulus decreased from that of the normal tissue in 8 weeks and then increased during the cholesterol feeding period. The global modulus did not change until 24 weeks and increased by 28 weeks. Histological observation revealed that the initial soft lesion was mainly composed of foam cells, and the stiffening accompanied first the appearance of smooth muscle cells in the top layer of the hyperplastic intima and then calcification in its bottom layer. The global elastic modulus did not change until marked calcification occurred in the tissue. These results suggest that change in mechanical properties of atherosclerotic lesion is not simple and has a close correlation with its histology. Assessment of local mechanical properties is important for studying mechanical properties of atherosclerotic arteries.

Contribution of actin filaments and microtubules to quasi-in situ tensile properties and internal force balance of cultured smooth muscle cells on a substrate

The effects of actin filaments (AFs) and microtubules (MTs) on quasi-in situ tensile properties and intracellular force balance were studied in cultured rat aortic smooth muscle cells (SMCs). A SMC cultured on substrates was held using a pair of micropipettes, gradually detached from the substrate while maintaining in situ cell shape and cytoskeletal integrity, and then stretched up to approximately 15% and unloaded three times at the rate of 1 mum every 5 s. Cell stiffness was approximately 20 nN per percent strain in the untreated case and decreased by approximately 65% and approximately 30% following AF and MT disruption, respectively. MT augmentation did not affect cell stiffness significantly. The roles of AFs and MTs in resisting cell stretching and shortening were assessed using the area retraction of the cell upon noninvasive detachment from thermoresponsive gelatin-coated dishes. The retraction was approximately 40% in untreated cells, while in AF-disrupted cells it was <20%. The retraction increased by approximately 50% and decreased by approximately 30% following MT disruption and augmentation, respectively, suggesting that MTs resist intercellular tension generated by AFs. Three-dimensional measurements of cell morphology using confocal microscopy revealed that the cell volume remained unchanged following drug treatment. A concomitant increase in cell height and decrease in cell area was observed following AF disruption and MT augmentation. In contrast, MT disruption significantly reduced the cell height. These results indicate that both AFs and MTs play crucial roles in maintaining whole cell mechanical properties of SMCs, and that while AFs act as an internal tension generator, MTs act as a tension reducer, and these contribute to intracellular force balance three dimensionally.

Haemodynamically dependent valvulogenesis of zebrafish heart is mediated by flow-dependent expression of miR-21

Heartbeat is required for normal development of the heart, and perturbation of intracardiac flow leads to morphological defects resembling congenital heart diseases. These observations implicate intracardiac haemodynamics in cardiogenesis, but the signalling cascades connecting physical forces, gene expression and morphogenesis are largely unknown. Here we use a zebrafish model to show that the microRNA, miR-21, is crucial for regulation of heart valve formation. Expression of miR-21 is rapidly switched on and off by blood flow. Vasoconstriction and increasing shear stress induce ectopic expression of miR-21 in the head vasculature and heart. Flow-dependent expression of mir-21 governs valvulogenesis by regulating the expression of the same targets as mouse/human miR-21 (sprouty, pdcd4, ptenb) and induces cell proliferation in the valve-forming endocardium at constrictions in the heart tube where shear stress is highest. We conclude that miR-21 is a central component of a flow-controlled mechanotransduction system in a physicogenetic regulatory loop.

RESIDUAL STRAIN AND LOCAL STRAIN DISTRIBUTIONS IN THE RABBIT ATHEROSCLEROTIC AORTA

Effect of atherosclerosis on the residual strain in the arterial wall was studied in the rabbit thoracic aorta. Atherosclerosis was induced by feeding a cholesterol diet or by combining denudation of aortic endothelial cells with the diet feeding. Diameter of each aorta in the physiological state was determined from a static pressure-diameter test. A ring specimen was obtained from the position where the diameter was measured. After the measurement of its no-load diameters, the ring was cut radially to measure the opening angle. Histological section was then obtained from the opened-up ring specimen. The section was divided into 32 subsections and local dimensions were measured for each subsection. Residual and in vivo strains referring to the opened-up configuration were calculated from the local dimensions and the diameters in the unloaded and physiological states. The opening angle significantly correlated with the area fraction of intimal hyperplasia until severe calcification occurred, while it had a negative correlation with the area fraction of calcified region. Histological observation of opened-up ring specimens indicated that the media adjacent to hyperplastic intima remained stretched, while that away from the intima relaxed. Analysis of local strains showed that, in the atherosclerotic aorta without calcification, higher residual strain (compressive), i.e., lower in vivo strain (tensile), appears in the thicker intima. These results suggest that: (a) intimal hyperplasia increases residual strain and thus reduces the in vivo strain exerted in the intima and (b) calcified tissue restrains the deformation of adjacent tissues. The intimal hyperplasia and the calcification may complicate strain fields in the atherosclerotic aorta.

Tensile properties of vascular smooth muscle cells: Bridging vascular and cellular biomechanics

Vascular walls change their dimensions and mechanical properties adaptively in response to blood pressure. Because these responses are driven by the smooth muscle cells (SMCs) in the media, a detailed understanding of the mechanical environment of the SMCs should reveal the mechanism of the adaptation. As the mechanical properties of the media are highly heterogeneous at the microscopic level, the mechanical properties of the cells should be measured directly. The tensile properties of SMCs are, thus, important to reveal the microscopic mechanical environment in vascular tissues; their tensile properties have a close correlation with the distribution and arrangement of elements of the cytoskeletal networks, such as stress fibers and microtubules. In this review, we first introduce the experimental techniques used for tensile testing and discuss the various factors affecting the tensile properties of vascular SMCs. Cytoskeletal networks are particularly important for the mechanical properties of a cell and its mechanism of mechanotransduction; thus, the mechanical properties of cytoskeletal filaments and their effects on whole-cell mechanical properties are discussed with special attention to the balance of intracellular forces among the intracellular components that determines the force applied to each element of the cytoskeletal filaments, which is the key to revealing the mechanotransduction events regulating mechanical adaptation. Lastly, we suggest future directions to connect tissue and cell mechanics and to elucidate the mechanism of mechanical adaptation, one of the key issues of cardiovascular solid biomechanics.

Heartbeat regulates cardiogenesis by suppressing retinoic acid signaling via expression of miR-143.

Cardiogenesis proceeds with concomitant changes in hemodynamics to accommodate the circulatory demands of developing organs and tissues. In adults, circulatory adaptation is critical for the homeostatic regulation of blood circulation. In these hemodynamics-dependent processes of morphogenesis and adaptation, a mechanotransduction pathway, which converts mechanical stimuli into biological outputs, plays an essential role, although its molecular nature is largely unknown. Here, we report that expression of zebrafish miR-143 is dependent on heartbeat. Knocking-down miR-143 results in de-repression of retinoic acid signaling, and produces abnormalities in the outflow tracts and ventricles. Our data uncover a novel epigenetic link between heartbeat and cardiac development, with miR-143 as an essential component of the mechanotransduction cascade.

Mechanical and dimensional adaptation of rabbit carotid artery culturedin vitro

The effects of the mechanical environment on arterial walls were investigated in rabbit common carotid arteries, cultured for six days under three different intraluminal pressures (0, 80 and 160 mmHg) in a perfusion culture system. The mechanical responses following the culture were examined using a quasi-static pressure-diameter test. Specimen viability was determined by smooth muscle contraction induced with KCl. Eighteen out of 21 cultured segments showed a peak reduction in diameter of more than 10% and were used for the analysis. The arterial segments cultured at 0 mmHg had a significantly smaller diameter than those cultured at other pressures. The segments cultured at higher pressure had lower incremental elastic moduli at 20 and 80 mmHg and higher moduli at 160 mmHg. The walls of the cultured segments were thicker in groups with higher pressure. These results indicate that, even in culture, the mechanical environment is a major determinant for the mechanical property and dimensions of the arterial wall. Arterial walls may respond to their mechanical environment even if other factors, such as hormonal environment and nervous stimuli, are kept unchanged.

Heterogeneous response of traction force at focal adhesions of vascular smooth muscle cells subjected to macroscopic stretch on a micropillar substrate

Traction force generated at focal adhesions (FAs) of cells plays an essential role in regulating cellular functions. However, little is known about how the traction force at each FA changes during cell stretching. Here we investigated dynamic changes in traction force at FAs during macroscopic stretching of porcine aortic smooth muscle cells (SMCs) cultured on elastic micropillar substrates. SMCs were cultured on polydimethylsiloxane (PDMS)-based substrates with a micropillar array, and stretched approximately in the direction of their major axis and then released by stretching and relaxing the substrates. This stretch-release cycle was repeated twice with cell strain rates of 0.3%/15s up to a 3% strain, and the deflection of the PDMS micropillars was measured simultaneously to obtain the traction force at each FA F, total force in the cells major axis direction F(all), and whole-cell strain ε(cell). Traction forces of SMCs during stretching varied widely with location: their changes at some pillars synchronized well with the applied strain ε(cell), but others did not synchronized. Whole-cell stiffness estimated as the slope of the loading limb of the F(all)-ε(cell) curves was ∼10nN/%, which was the same order of magnitude of the reported stiffness of cultured SMCs obtained in a tensile test. Interestingly, F(all) at a zero-strain state (pretension at the whole-cell level) actively increased in some cells following the loading/unloading process, as did whole-cell stiffness. Such a change did not occur in cultured SMCs in the tensile test in which cells were held with a pair of micropipettes coated with nonspecific adhesive. These results indicate that SMCs showed a myogenic response when stretched through their multiple FAs, but not through nonspecific adhesions on their membrane. SMCs may behave differently depending on the sites through which they are stretched.