Stress Fiber Orientation Research Articles

103 培養内皮細胞の変形に基づくストレスファイバーの繰返し変形下での配向に関する理論的検討

103 培養内皮細胞の変形に基づくストレスファイバーの繰返し変形下での配向に関する理論的検討

A202 培養内皮細胞ストレスファイバーの変形限度に基づいた繰返し変形下での 3 次元的配向の予測

A202 培養内皮細胞ストレスファイバーの変形限度に基づいた繰返し変形下での 3 次元的配向の予測

Numerical Simulation of Stress Fiber Orientation in Cultured Endothelial Cells under Biaxial Cyclic Deformation Using the Strain Limit Hypothesis.

This paper verifies the hypothesis that a stress fiber (SF) will only be oriented in the direction in which the fiber strain does not exceed a limit for the maximally deformed state of the substrate. We assume three-dimensional and uniform deformation of a cell on a substrate and predict the three-dimensional orientations of the SFs during cyclic deformation tests of the substrate. These predictions are in good agreement with experimental data for the basal membrane under simple cyclic elongations, and reproduce an observation of the three-dimensional orientation of actin filaments under equibiaxial cyclic stretching. Results from a stress analysis using a finite element model of an adherent cell with a nucleus show that a tensile stress develops in the direction transverse to the stretch axis under pure uniaxial stretching. This indicates that SFs are oriented to minimize strain, not the stress, and that a nucleus complicates the stress distribution in a cell.

Application of a Compliance Method to Fatigue Crack Growth Analysis of GLARE3 Fiber/Metal Laminates.

The objectives of this study were to investigate the effectiveness of a compliance method for analyzing the fatigue crack growth of GLARE 3 fiber/metal laminates, and to ascertain the influence of specimen thickness, notch length, and fiber orientation upon fatigue crack growth. The materials tested were GLARE 3-5/4 and GLARE 3-3/2. Centrally notched specimens with two kinds of notch length and two kinds of fiber orientation were fatigue tested under constant amplitude loading. The experimental stress intensity factors, Kexp, for the 2024-T3 aluminum-alloy layers of a GLARE 3 were obtained from the relationship between crack length and specimen compliance. The test results clarified that da/dN-ΔKexp relationships are roughly independent of maximum stress level, specimen thickness, notch length, and fiber orientations and that the compliance method is effective for analyzing fatigue crack growth in GLARE 3 laminates.

Flow-Induced Cytoskeletal Changes in Endothelial Cells Growing on Curved Surfaces

Objective: Our purpose was to investigate the effect of the shape of the growth surface (curved versus flat) on flow-induced F-actin organization in endothelial cells. Methods: Human umbilical vein endothelial cells were grown to confluence on curved or flat surfaces. Microchannels (curved surface, 10- to 30-µm radius) or parallel plate flow chambers were perfused (30 minutes to 6 hours) at physiological flow rates (wall shear stress 1 to 10 dyn/cm2). Results: On curved surfaces, the number of central F-actin stress fibers (for cells of equal area) decreased from 4.8 ± 0.3 (mean ± SE, n = 36) (static) to 0.9 ± 0.5 per cell in perfused microchannels. Perfusion with 100 µM histamine prevented this response to flow (5.5 ± 0.8 per cell, n = 12). Stress fibers were initially aligned with the long axis of the microchannel at an angle of 9 ± 0.7° (static). With flow, alignment of the few remaining central F-actin stress fibers with respect to the long axis of the microchannel decreased to 19 ± 4°; this was prevented by perfusion with histamine (5.6 ± 1°). The number of stress fibers per cell, for cells grown on flat surfaces (8.1 ± 0.3, static, n = 36) was significantly greater than for cells on curved surfaces, and did not change with flow (8.1 ± 0.5 per cell, n = 6). On flat surfaces, the stress fiber orientation (with respect to the longitudinal axis of the channel) was 42 ± 1.4° (static) and did not change with flow (38 ± 4.2°). Conclusions: Endothelial cells on curved growth surfaces respond to flow rapidly, with marked changes in F-actin central stress fiber formation. This implicates a tight relationship between cell shape and the environmental substrate, and suggests that the shape of the endothelial cell significantly impacts its ability to respond to its environment.

Flow‐Induced Cytoskeletal Changes in Endothelial Cells Growing on Curved Surfaces

Our purpose was to investigate the effect of the shape of the growth surface (curved versus flat) on flow-induced F-actin organization in endothelial cells. Human umbilical vein endothelial cells were grown to confluence on curved or flat surfaces. Microchannels (curved surface, 10- to 30-microm radius) or parallel plate flow chambers were perfused (30 minutes to 6 hours) at physiological flow rates (wall shear stress 1 to 10 (dyn/cm2). On curved surfaces, the number of central F-actin stress fibers (for cells of equal area) decreased from 4.8+/-0.3 (mean +/- SE, n = 36) (static) to 0.9+/-0.5 per cell in perfused microchannels. Perfusion with 100 microM histamine prevented this response to flow (5.5+/-0.8 per cell, n = 12). Stress fibers were initially aligned with tile long axis of the microchannel at an angle of 9+/-0.7 degrees (static). With flow, alignment of the few remaining central F-actin stress fibers with respect to the long axis of the microchannel decreased to 19+/-4 degrees this was prevented by perfusion with histamine (5.6+/-1 degrees). The number of stress fibers per cell, for cells grown on flat surfaces (8.1+/-0.3, static, n = 36) was significantly greater than cells on curved surfaces, and did not change with flow (8.1+/-0.5 per cell, n = 6). On flat surfaces, the stress fiber orientation with respect to the longitudinal axis of the channel) was 42+/-1.4 degrees (static) and did not change with flow (38+/-4.2 degrees). Endothelial cells on curved growth surfaces respond to flow rapidly, with marked changes in F-actin central stress fiber formation. This implicates a tight relationship between cell shape and the environmental substrate, and suggests that the shape of the endothelial cell significantly impacts its ability to respond to its environment.

Contractility affects stress fiber remodeling and reorientation of endothelial cells subjected to cyclic mechanical stretching.

We studied the effect of contractility on stress fiber remodeling and orientation response of human aortic endothelial cells subjected to cyclic mechanical stretching. The cells were grown on silicone membranes subjected to 10% cyclic pure uniaxial stretching in the presence or absence of 2,3 butanedione monoxime (BDM), a proven inhibitor of cellular contractility. It was found that treatment of the cells with BDM (40 mM) abolished stress fibers and blocked cell reorientation in response to cyclic stretching, indicating that contractility is required for these two cellular responses. When cells were stretched in the presence of N-acetylcysteine (NAG, 20 mM), a hydrogen peroxide (H2O2) scavenger, stress fibers were still formed and the cells reoriented--but more slowly. Specifically, compared with untreated cells, NAG treated cells after 0.5, 1, and 3 h of 10% stretching had significantly (p<0.005) less skewed orientation distributions than those of untreated cells. After the cells were treated with both NAG (20 mM) and nordihydroguaiaretic acid (NDGA, 50 microM), another antioxidant, however, stress fibers were abolished and cell reorientation was completely blocked. These results indicate that reactive oxygen species (ROS), including H2O2, affect stress fiber remodeling and reorientation of endothelial cells in response to cyclic stretching. We suggest that the effect of ROS on stress fiber remodeling and cell reorientation is due to the ability of ROS to regulate cellular contractility, which is crucial for these cellular responses.

Theoretical study of intracellular stress fiber orientation under cyclic deformation

We studied stress fiber orientation under a wide range of uniaxial cyclic deformations. We devised and validated a hypothesis consisting of two parts, as follows: (1) a stress fiber aligns to avoid a mechanical stimulus in the fiber direction under cyclic deformation. This means that, among all allowable directions, a stress fiber aligns in the direction which minimizes the stimulus, i.e., the summation of the changes in length of the stress fiber over one stretch cycle; and (2) there is a limit in the sensitivity of the cellular response to the mechanical stimulus. Due to this sensing limit, the orientation angle in stress fibers is distributed around the angle corresponding to the minimum stimulus. To validate this hypothesis, we approximated an anisotropic deformation of the membrane on which cells were to be cultured. We then obtained the relationships between the stretch range and the fiber angle in the undeformed state which minimize the mechanical stimuli, assuming that the membrane on which stress fibers and cells adhered was homogeneous and incompressible. Numerical simulation results showed that the proposed hypothesis described our previous experimental results well and was consistent with the experimental results in the literature. The simulation results, taking account of the second part of the hypothesis with a small value for the limit in sensitivity to the mechanical stimulus, could explain why cell orientation is distributed so widely with cyclic stretch ranges of <10%. The proposed hypothesis can be applied to various types of deformation because the mechanical stimulus is always sensed and accumulates under cyclic deformation without the necessity of a reference state to measure the stimulus.

Flow-Induced Cytoskeletal Changes in Endothelial Cells Growing on Curved Surfaces

Objective: Our purpose was to investigate the effect of the shape of the growth surface (curved versus flat) on flow-induced F-actin organization in endothelial cells. Methods: Human umbilical vein endothelial cells were grown to confluence on curved or flat surfaces. Microchannels (curved surface, 10- to 30-µm radius) or parallel plate flow chambers were perfused (30 minutes to 6 hours) at physiological flow rates (wall shear stress 1 to 10 dyn/cm2). Results: On curved surfaces, the number of central F-actin stress fibers (for cells of equal area) decreased from 4.8 ± 0.3 (mean ± SE, n = 36) (static) to 0.9 ± 0.5 per cell in perfused microchannels. Perfusion with 100 µM histamine prevented this response to flow (5.5 ± 0.8 per cell, n = 12). Stress fibers were initially aligned with the long axis of the microchannel at an angle of 9 ± 0.7° (static). With flow, alignment of the few remaining central F-actin stress fibers with respect to the long axis of the microchannel decreased to 19 ± 4°; this was prevented by perfusion with histamine (5.6 ± 1°). The number of stress fibers per cell, for cells grown on flat surfaces (8.1 ± 0.3, static, n = 36) was significantly greater than for cells on curved surfaces, and did not change with flow (8.1 ± 0.5 per cell, n = 6). On flat surfaces, the stress fiber orientation (with respect to the longitudinal axis of the channel) was 42 ± 1.4° (static) and did not change with flow (38 ± 4.2°). Conclusions: Endothelial cells on curved growth surfaces respond to flow rapidly, with marked changes in F-actin central stress fiber formation. This implicates a tight relationship between cell shape and the environmental substrate, and suggests that the shape of the endothelial cell significantly impacts its ability to respond to its environment.

1415 単軸繰返し負荷下での培養血管内皮細胞ストレスファイバーの配向に関する理論的予測

1415 単軸繰返し負荷下での培養血管内皮細胞ストレスファイバーの配向に関する理論的予測

Assessment of stress fiber orientation during healing of radial keratotomy wounds using confocal microscopy.

Radial keratotomy (RK) is a refractive surgical procedure in which partial thickness incisions are made in the cornea in order to alter its shape. Previous studies suggest that RK wounds undergo changes in wound gape in response to the ingrowth of myofibroblasts which mediate subsequent wound contraction and may modulate changes in corneal curvature seen after RK. A recent quantitative analysis of f-actin organization in full-thickness incisional wounds in the rabbit demonstrated that microfilament bundles (stress fibers) present in myofibroblasts align parallel to the long axis of the wound during wound contraction. To investigate whether the same pattern of alignment occurs after RK, a similar analysis of f-actin organization was undertaken using the cat RK model. Radial keratotomy wounds were studied from 10 to 28 days after surgery using en block staining with fluorescein isothiocyanate (FITC) phalloidin, and three-dimensional (3-D) datasets (z-series of en face optical sections) were collected using laser confocal microscopy at various regions within the wound. In addition, conventional en face sections were double-labeled using combinations of phalloidin and antibodies to fibronectin and alpha 5 beta 1 integrin. Myofibroblast ingrowth started in the bottom of the wound and progressed anteriorly. At 10 to 14 days, f-actin was predominantly distributed in long, thick bundles (stress fibers) within the wound. These fibers appeared to be randomly oriented anteriorly, but became progressively more aligned with the long axis of the wound posteriorly. At 21 days, the stress fibers were predominantly oriented parallel to the long axis of the wound at all levels. F-actin, fibronectin and integrin were coaligned at both the 14 and 21 day time points. Since the majority of wound closure occurs between 14 and 28 days after surgery, we conclude that parallel alignment of the actin filament-fibronectin-integrin assembly in the cat RK model is associated with wound contraction.

The Change of F-actin Distribution and Morphology of Cultured Bovine Aortic Endothelial Cells in the Early Stage of Fluid Shear Stress Exposure.

The time course changes of F-actin distribution and morphology of cultured bovine aortic endothelial cells under shear flow conditions were analyzed. Fully confluent endothelial cells cultured on glass were subjected to fluid shear stress of 2 Pa. Experiments were performed for one-way flow for 20, 40 min, 1, 3 and 6 hours. After the flow exposure F-actin filaments were stained with rhodamine-phalloidin. The morphological parameters of endothelial cells (shape index and angle of cell orientation), the appearance frequency of the stress fibers and the angle of stress fiber orientation were measured. F-actin filaments were mainly localized at periphery of the cells, although some filaments were seen as star-like configurations in the center portion under no-flow conditions. In some cells (16%) exposed to shear stress for 20 min, stress fibers which were aligned with cell axes were partly observed. In this stage, however, cell morphological changes were not observed. In many cells (64%) exposed to shear stress for 1 hour, the star-like structure of filaments disappeared in the center portion, and stress fibers appeared. After 6 hours, stress fibers were observed in the most cells (91%) and began to align with flow direction. The cells in which stress fibers appeared significantly elongated, but the cells without stress fibers had kept their polygonal shape. From these results, it is expected that F-actin filaments may play a major role for sensing the level and direction of shear stress and for changing morphology of endothelial cells.

Quantitative analysis of stress fiber orientation during corneal wound contraction

Previous studies of actin and actin-binding proteins in corneal myofibroblasts suggest the development of a contractile apparatus composed, in part, of F-actin micro-filament bundles, i.e. stress fibers. To better understand the mechanics of wound contraction and the relationship between microfilament bundles and wound closure, we have analyzed the spatial and temporal organization of stress fibers during the process of corneal wound healing. Rabbit corneas (26 eyes) received 6 mm full-thickness, central incisions and were studied at various times for F-actin organization using en bloc (whole cornea) staining with FITC-phalloidin, as well as conventional histological techniques. 3-D datasets (z-series of 40 en face optical sections, 1 micron steps) were collected using the Biorad MRC-600 laser scanning confocal microscope at various regions within the wound. At 7 days, 3-D analysis showed randomly oriented, interconnected F-actin filament bundles (stress fibers). Between 7 and 28 days, stress fibers appeared to organize gradually into planes parallel to the wound surface, with a large population achieving a final orientation nearly parallel to the long axis of the wound. Using Fourier Transform analysis techniques, an orientation index (OI) was calculated to quantitate global fiber orientation at each time point. Analysis of variance demonstrated a significant change (P < 0.001) in overall stress fiber orientation from a random distribution at day 7 to an alignment more parallel to the lateral wound borders at day 28. Overall, these data suggest that stress fibers undergo temporal changes in spatial organization that correlate with wound closure, and that wound closure does not involve the development of previously described contractile or tractional forces aligned directly across the wound.

レーザ走査共焦点顕微鏡による局所血流構造が血管内皮細胞の構造及び機能に与える影響の解析

The blood velocity pattern near the cranial wall at the renal ostia, where atherosclerotic lesions are prone to develop, are characterized by 1) slow time-averaged shear rate, 2) separation of the flow, and 3) a time-varying oscillation of the flow (Arterioscler Thromb 1992; 12: 626-632). To elucidate the relation between the velocity pattern and localization of development of atherosclerosis, we analyzed the fine structure and function of the vascular wall by using laser scanning confocal microscope. In a Wistar rat the renal artery was perfusion fixed at a pressure of 75 mmHg. F-actin and DNA in nuclei were stained with rhodamine phalloidin and bisBenzimide, respectively. Low density lipoprotein (LDL) scavenger transport was investigated by injection of DiI-Ac-LDL before sacrificed. At the cranial wall, where atherosclerotic lesions as prone to develop, the shapes of endothelial cells and nuclei were relatively round, comparing to the caudal wall or the distal portion of renal artery, where atherosclerotic lesions hardly develop. The orientation of nuclei and stress fibers at the cranial wall was relatively random. With ten minutes incubation, Ac-LDL were observed in the endothelial cell and smooth muscle cell with higher density in the former. The density of Ac-LDL in vascular media at the cranial wall was much higher than that the caudal wall. In conclusion, the velocity pattern localizes the development of atherosclerosis via the change in the endothelial cell structure and Ac-LDL transport within the vascular wall.

Expression of stress fibers in bullfrog mesothelial cells in response to tension

The relationship between stress fibers and tension in mesothelial cells of the bullfrog small intestine was examined by fluorescence cytochemistry using en face mesothelial cell preparations. In nontreated controls, actin revealed by rhodamine-phalloidin staining was localized only along the margins of the mesothelial cells. On the other hand, many stress fibers were formed in the mesothelial cells within 5–7 min after stretching of the intestinal wall in a given direction. The orientation of stress fibers within the cells was coincident with the direction of the tension applied. These cytoplasmic fibers disappeared almost completely from the mesothelial cells within 30 min after the release of tension. According to a difference in the intensity of tension necessary for stress fiber expression, the intestinal mesothelial cells were classified into two groups. Furthermore, cells containing stress fibers in each group showed a rapid increase in number once a given value of tension was applied. The present results indicate that the mesothelial cells of bullfrog small intestine may develop stress fibers to counteract tension exerted on the intestinal wall. Such stress fibers may serve to maintain cellular integrity by strengthening the cellular attachment to subepithelial tissue.

Effects of fiber orientation on the stress distribution in model composites

AbstractA Raman‐mechanical technique was used to study the relationship between stress distribution and fiber orientation in model composites. Our experimental data generally were consistent with most simplistic mechanical models. A more complete analysis, using the Eshelby equivalent inclusion method, fitted our experimental data exceptionally well. For large applied strains, compressive failure occurred for fibers which were oriented at high angles relative to the draw direction. This occurred because of the lateral shrinkage associated with the matrix when the sample was stretched. The effect of fiber end geometry on the stress distribution for these misaligned fibers was the same as observed earlier. Tapered‐end fibers generally carried loads more efficiently in composites than blunt‐end fibers.

Organization of actin cytoskeleton in normal and regenerating arterial endothelial cells.

The distribution of actin stress fibers in normal and regenerating (after endothelial denudation by means of a balloon catheter) rabbit aortic endothelial cells has been studied by means of immunofluorescence with human actin autoantibodies on en face endothelial cell preparations. Our results show that: (i) under normal conditions actin is accumulated as a network at the periphery of endothelial cells. Stress fibers are present only in endothelial cells located immediately below intercostal artery branches; (ii) stress fibers develop in endothelial cells early during regeneration and persist after the end of endothelial mitotic and motile activities; and (iii) the orientation of stress fibers within the cytoplasm follows the direction of blood flow, with the exception of stress fibers situated in cells at the edge of the wound, when endothelial cell progression toward the denuded area as well as mitotic activity have ceased. We conclude that stress fibers are an organelle present in endothelial cells in vivo and that they reorganize during endothelial cell adaptation to unfavorable or pathological situations.