Elastin and collagen fibers play important roles in the mechanical properties of aortic media. Because knowledge of local fiber structures is required for detailed analysis of blood vessel wall ...mechanics, we investigated 3D microstructures of elastin and collagen fibers in thoracic aortas and monitored changes during pressurization. Using multiphoton microscopy, autofluorescence images from elastin and second harmonic generation signals from collagen were acquired in media from rabbit thoracic aortas that were stretched biaxially to restore physiological dimensions. Both elastin and collagen fibers were observed in all longitudinal–circumferential plane images, whereas alternate bright and dark layers were observed along the radial direction and were recognized as elastic laminas (ELs) and smooth muscle-rich layers (SMLs), respectively. Elastin and collagen fibers are mainly oriented in the circumferential direction, and waviness of collagen fibers was significantly higher than that of elastin fibers. Collagen fibers were more undulated in longitudinal than in radial direction, whereas undulation of elastin fibers was equibiaxial. Changes in waviness of collagen fibers during pressurization were then evaluated using 2-dimensional fast Fourier transform in mouse aortas, and indices of waviness of collagen fibers decreased with increases in intraluminal pressure. These indices also showed that collagen fibers in SMLs became straight at lower intraluminal pressures than those in EL, indicating that SMLs stretched more than ELs. These results indicate that deformation of the aorta due to pressurization is complicated because of the heterogeneity of tissue layers and differences in elastic properties of ELs, SMLs, and surrounding collagen and elastin.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Understanding in multicellular behaviors in three-dimensional (3D) culture models such as organoids is important to help us better comprehend the mechanisms of the morphogenesis and functions of ...diverse organs in vivo cellular environment. In this study, we elucidated the multicellular behaviors of the osteocytic spheroids in response to the chemically induced osteogenesis supplements (OS). Particularly, we conducted 1) size change measurement, 2) fusion experiment, and 3) collagen embedding experiment of spheroids, in response to the OS. We found out that the OS alters the multicellular behaviors of the spheroid by greater reduction in the size change measurement and slowing down the speed of fusion experiment and collagen embedding experiment of the spheroids. We also highlighted that the driving force of these changes was the tight actin filaments generated on the surface of the spheroids. Hence, the results altogether indicate that the spheroid model exerted the different multicellular behaviors against the differentiation capability. This study will contribute to understanding the multicellular behaviors of the 3D culture model reconstructed by the cells with greater cell-cell interaction force.
•Chemical osteogenesis supplements modulated multicellular behavior of the spheroid.•Osteogenesis supplements enhanced actin filaments on the surface of the spheroid.•Tight actin filaments deformed the nuclei on the surface of the spheroid.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract 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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The media of aortic wall is characterized by altering layers of elastin and smooth muscle cells (SMCs), along with collagen fibers in both layers, and plays a central role in functional and ...pathological remodeling such as hypertension and atherosclerosis. Because the arterial function is linked closely to the arterial wall internal structure, it is essential to investigate the alteration of the arterial microstructure during macroscopic deformation to understand cardiovascular pathologies. The present study adopted a tissue clearing method in three-dimensional mechanical characterization of rat thoracic aorta, and successfully observed changes in the structure of each of the three primary components of the aorta under intraluminal pressurization while maintaining tissue mechanical integrity and flexibility. Layers of elastic fibers and SMCs deformed greater on the intimal side than those on the adventitial side. Furthermore, there was a structural agreement in the alignment angle between SMC nuclei and elastic fibers on their intimal side, but not on the adventitial side. This is the first study that changes in the microstructure of three primary components of the aorta were visualized and evaluated through the aorta. The method established here would also be useful to understand tissue mechanics of other load-bearing soft tissues.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Although the human papillomavirus (HPV) vaccine is effective for preventing cervical cancers, this vaccine does not eliminate pre‐existing infections, and alternative strategies have been warranted. ...Here, we report that FOXP4 is a new target molecule for differentiation therapy of cervical intraepithelial neoplasia (CIN). An immunohistochemical study showed that FOXP4 was expressed in columnar epithelial, reserve, and immature squamous cells, but not in mature squamous cells of the normal uterine cervix. In contrast with normal mature squamous cells, FOXP4 was expressed in atypical squamous cells in CIN and squamous cell carcinoma lesions. The FOXP4‐positive areas significantly increased according to the CIN stages from CIN1 to CIN3. In monolayer cultures, downregulation of FOXP4 attenuated proliferation and induced squamous differentiation in CIN1‐derived HPV 16‐positive W12 cells via an ELF3‐dependent pathway. In organotypic raft cultures, FOXP4‐downregulated W12 cells showed mature squamous phenotypes of CIN lesions. In human keratinocyte‐derived HaCaT cells, FOXP4 downregulation also induced squamous differentiation via an ELF3‐dependent pathway. These findings suggest that downregulation of FOXP4 inhibits cell proliferation and promotes the differentiation of atypical cells in CIN lesions. Based on these results, we propose that FOXP4 is a novel target molecule for nonsurgical CIN treatment that inhibits CIN progression by inducing squamous differentiation.
Downregulation of FOXP4 inhibits cell proliferation and promotes differentiation of atypical cells in CIN lesions. We propose that FOXP4 is a novel target molecule for non‐surgical CIN treatment that inhibits CIN progression by inducing squamous differentiation.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Osteogenic differentiation has been reportedly regulated by various mechanical stresses, including fluid shear stress and tensile and compressive loading. The promotion of osteoblastic ...differentiation by these mechanical stresses is accompanied by reorganization of the F-actin cytoskeleton, which is deeply involved in intracellular forces and the mechanical environment. However, there is limited information about the effect on the mechanical environment of the intracellular nucleus, such as the mechanical properties of the nucleus and intracellular forces exerted on the nucleus, which have recently been found to be directly involved in various cellular functions. Here, we investigated the changes in the intracellular force applied to the nucleus and the effect on nuclear morphology and mechanical properties during osteogenic differentiation in human osteoblast-like cells (Saos-2). We carried out cell morphological analyses with confocal fluorescence microscopy, nuclear indentation test with atomic force microscopy (AFM), and fluorescence recovery after photobleaching (FRAP) for intranuclear DNA. The results revealed that a significant reorganization of the F-actin cytoskeleton from the nuclear surfaces to the cell periphery occurred in the osteogenic differentiation processes, simultaneously with the reduction of compressive forces to the nucleus. Such changes also facilitated nuclear shrinkage and stiffening, and further intranuclear chromatin compaction. The results indicate that the reduction of the intracellular compressive force due to reorganization of the F-actin cytoskeleton affects the intra- and extra-mechanical environment of the nucleus, and this change may affect gene expression and DNA replication in the osteogenic differentiation process.
•Mechanical environment of cell nucleus during bone differentiation was investigated.•F-actin was concentrated at cell periphery to reinforce the cell-cell junctions.•The nuclear shrinkage and stiffening occurred in connection with intracellular force reduction.•The intranuclear chromatin compaction was facilitated in bone differentiation.•Intra- and extra-nuclear mechanics are deeply involved in bone differentiation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The deformation of the cell nucleus may cause dispersion of chromatin and eventually enhance transcription, translation, and protein expression. If this happens in the hypertensive artery, an ...excessive stretch of smooth muscle cell (SMC) nuclei caused by hypertension may provoke wall thickening. Here, we measured deformation of SMC nuclei in rabbit thoracic aortas stretched in different directions. Thin 0.2-mm-thick specimens were sliced in the direction perpendicular to their axial and circumferential directions, and stretched in the circumferential and axial directions, respectively. The deformation of the actin filament (AF) network was similar to that of the whole tissue, whereas the deformation of the nucleus was significantly smaller than the others. Notably, the nucleus seldom deformed when the tissue was stretched in the axial direction. A novel cell model in which the nucleus is connected to the extracellular matrix via the AF network successfully explained the relative unresponsiveness of the nucleus to the axial stretch. It has been pointed out that stress is maintained constant in the circumferential direction but not in the axial direction in the artery wall during hypertension. The relative unresponsiveness of the nucleus to the axial stretch represented in this study explains this phenomenon.
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•Microscopic deformation of aorta measured against circumferential and axial stretches.•Actin filament network deforms similarly to that of macroscopic stretches.•SMC nuclei deform less than macroscopic stretch especially in axial direction.•Tiny deformation of nuclei may explain insensitiveness of aortic wall to axial stress.•Simple cell model explaining anisotropic response of nuclear deformation was proposed.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
FRET-based sensors are utilized for real-time measurements of cellular tension. However, transfection of the sensor gene shows low efficacy and is only effective for a short period. Reporter mice ...expressing such sensors have been developed, but sensor fluorescence has not been measured successfully using conventional confocal microscopy. Therefore, methods for spatiotemporal measurement of cellular tension in vivo or ex vivo are still limited. We established a reporter mouse line expressing FRET-based actinin tension sensors consisting of EGFP as the donor and mCherry as the acceptor and whose FRET ratio change is observable with confocal microscopy. Tension-induced changes in FRET signals were monitored in the aorta and tail tendon fascicles, as well as aortic smooth muscle cells isolated from these mice. The pattern of FRET changes was distinctive, depending on tissue type. Indeed, aortic smooth muscle cells exhibit different sensitivity to macroscopic tensile strain in situ and in an isolated state. This mouse strain will enable novel types of biomechanical investigations of cell functions in important physiological events.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Biomechanics Laboratory, Department of Mechanical Engineering, Nagoya Institute of Technology, Nagoya, Japan
Submitted 18 February 2008
; accepted in final form 7 October 2008
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 15% and unloaded three times at the rate of 1 µm every 5 s. Cell stiffness was 20 nN per percent strain in the untreated case and decreased by 65% and 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 40% in untreated cells, while in AF-disrupted cells it was <20%. The retraction increased by 50% and decreased by 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.
cellular biomechanics; mechanical properties; hysteresis; cell retraction; cellular prestress
Address for reprint requests and other correspondence: K. Nagayama, Nagoya Institute of Technology Omohi College, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan (e-mail: k-nagaym{at}nitech.ac.jp )
Abstract Determination of the local amount and direction of collagen fibers during deformation is crucial for an understanding of the mechanical behavior of aortic tissues. Since most conventional ...methods cannot be used for this purpose, we propose a method to quantify the local amount and direction of fibers by simply measuring the optical properties of the specimen. After confirming the linear correlation between the retardance and thickness of sections of porcine thoracic aortas (PTAs) ranging from 15 to 300 μm, we investigated the effects of their structural components, i.e., smooth muscle cells (SMCs), elastin and collagen, on the retardance of whole tissues. Decellularization of SMCs did not change the retardance of PTA sections significantly. Patterns in autofluorescent and immunofluorescent images of elastin purified from bovine nuchal ligaments did not match those in retardance images. Images of collagen in PTA sections stained with picrosirius red were similar to corresponding retardance images. The slow axis azimuth corresponded to the circumferential direction of the aorta. Results indicate that collagen in aortas can be quantified by measuring the retardance and slow axis azimuth of whole aortic tissues. Application of this technique to PTAs showed that retardance was higher in dorsal and distal regions than ventral and proximal regions, respectively, indicating that the aortas contain more collagen in distal and dorsal regions than proximal and ventral regions, respectively. Both results were in accordance with previous findings. Measurement of retardance is useful to quantify the amount of collagen in unfixed aortas.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
