Polarized light retardation analysis allows for the evaluation of tension in individual stress fibers.

The tension in the stress fibers (SFs) of cells plays a pivotal role in determining biological processes such as cell migration, morphological formation, and protein synthesis. Our previous research developed a method to evaluate the cellular contraction force generated in SFs based on photoelasticity-associated retardation of polarized light; however, we employed live cells, which could have caused an increase in retardation and not contraction force. Therefore, the present study aimed to confirm that polarized light retardation increases inherently due to contraction, regardless of cell activity. We also explored the reason why retardation increased with SF contractions. We used SFs physically isolated from vascular smooth muscle cells to stop cell activity. The retardation of SFs was measured after ATP administration, responsible for contracting SFs. The SFs were imaged under optical and electron microscopes to measure SF length, width, and retardation. The retardation of isolated SFs after ATP administration was significantly higher than before. Thus, we confirmed that retardation increased with elevated tension in individual SFs. Furthermore, the SF diameter decreased while the SF length remained almost constant. Thus, we conclude that a contraction force-driven increase in the density of SFs is the main factor for the rise in polarized light retardation.

Decoding the Effect of Hydrostatic Pressure on TRPV1 Lower-Gate Conformation by Molecular-Dynamics Simulation.

In response to hydrostatic pressure, the cation channel transient receptor potential vanilloid 1 (TRPV1) is essential in signaling pathways linked to glaucoma. When activated, TRPV1 undergoes a gating transition from a closed to an open state that allows the influx of Ca2+ ions. However, the gating mechanism of TRPV1 in response to hydrostatic pressure at the molecular level is still lacking. To understand the effect of hydrostatic pressure on the activation of TRPV1, we conducted molecular-dynamics (MD) simulations on TRPV1 under different hydrostatic pressure configurations, with and without a cell membrane. The TRPV1 membrane-embedded model is more stable than the TPRV1-only model, indicating the importance of including the cell membrane in MD simulation. Under elevated pressure at 27.6 mmHg, we observed a more dynamic and outward motion of the TRPV1 domains in the lower-gate area than in the simulation under normal pressure at 12.6 mmHg. While a complete closed-to-open-gate transition was not evident in the limited course of our MD simulations, an increase in the channel radius at the lower gate was observed at 27.6 mmHg versus that at 12.6 mmHg. These findings provide novel information regarding the effect of hydrostatic pressure on TRPV1 channels.

Comparison of the histology and stiffness of ventricles in Anura of different habitats.

Vertebrate hearts have undergone marked morphological and structural changes to adapt to different environments and lifestyles as part of the evolutionary process. Amphibians were the first vertebrates to migrate to land. Transition from aquatic to terrestrial environments required the ability to circulate blood against the force of gravity. In this study, we investigated the passive mechanical properties and histology of the ventricles of three species of Anura (frogs and toads) from different habitats, Xenopus laevis (aquatic), Pelophylax nigromaculatus (semiaquatic), and Bufo japonicus formosus (terrestrial). Pressure-loading tests demonstrated stiffer ventricles of P. nigromaculatus and B. j. formosus compared X. laevis ventricles. Histological analysis revealed a remarkable difference in the structure of cardiac tissue: thickening of the compact myocardium layer of P. nigromaculatus and B. j. formosus and enrichment of the collagen fibers of B. j. formosus. The amount of collagen fibers differed among the species, as quantitatively confirmed by second-harmonic generation light microscopy. No significant difference was observed in cardiomyocytes isolated from each animal, and the sarcomere length was almost the same. The results indicate that the ventricles of Anura stiffen during adaptation to life on land.

Direct application of mechanical stimulation to cell adhesion sites using a novel magnetic-driven micropillar substrate.

Cells change the traction forces generated at their adhesion sites, and these forces play essential roles in regulating various cellular functions. Here, we developed a novel magnetic-driven micropillar array PDMS substrate that can be used for the mechanical stimulation to cellular adhesion sites and for the measurement of associated cellular traction forces. The diameter, length, and center-to-center spacing of the micropillars were 3, 9, and 9 µm, respectively. Sufficient quantities of iron particles were successfully embedded into the micropillars, enabling the pillars to bend in response to an external magnetic field. We established two methods to apply magnetic fields to the micropillars (Suresh 2007). Applying a uniform magnetic field of 0.3 T bent all of the pillars by ~4 µm (Satcher et al. 1997). Creating a magnetic field gradient in the vicinity of the substrate generated a well-defined local force on the pillars. Deflection of the micropillars allowed transfer of external forces to the actin cytoskeleton through adhesion sites formed on the pillar top. Using the magnetic field gradient method, we measured the traction force changes in cultured vascular smooth muscle cells (SMCs) after local cyclic stretch stimulation at one edge of the cells. We found that the responses of SMCs were quite different from cell to cell, and elongated cells with larger pre-tension exhibited significant retraction following stretch stimulation. Our magnetic-driven micropillar substrate should be useful in investigating cellular mechanotransduction mechanisms.

Measurement of surface topography and stiffness distribution on cross-section of Xenopus laevis tailbud for estimation of mechanical environment in embryo.

The stress distribution inside a Xenopus laevis tailbud embryo was estimated to examine the cause of the straightening and elongation. The embryos were cut in the middle, yielding a cross-section perpendicular to the body axis. The section was not flat, owing to the residual stress relief. The stress needed to restore the flatness corresponded to the stress inside the embryo and was calculated using the surface topography and Young's-moduli in the section. We found the areas of the notochord (Nc), neural tube (NT), and abdominal tissue (AT) bulged in the cross-section, which revealed that compressive forces acted in these tissues. The moduli of the Nc, NT, and AT were in the order of several thousand, hundred, and tens of pascals, respectively. In the Nc, the compressive force was largest and increased with the development, suggesting Nc playing a central role in the elongation. The bending moment generated by the AT was 10 times higher than that by the Nc in the early stages of the tailbud formation, and the two were similar in the latter stages, suggesting that the compressive force in the AT was the major cause of the straightening during the early stage. The straightening and elongation could be orchestrated by changes in the compressive forces acting on the Nc, NT, and AT over time. For the sake of simplicity, we calculated the compressive force only and neglected the tensile force. Thus, it should be noted that the amount of the compressive force was somewhat overestimated.

Predictive Methods for Thrombus Formation in the Treatment of Aortic Dissection and Cerebral Aneurysms: A Comprehensive Review.

Thrombus formation plays a crucial role in the clinical treatment of certain diseases. In conditions such as aortic dissection and cerebral aneurysm, complete thrombus occlusion in the affected region is desired to reduce blood flow into the false lumen or aneurysm sac, leading to a decrease in the tension exerted on the vascular wall and making it less likely to rupture. However, desired thrombosis sometimes fails to occur. Predicting thrombus formation can provide valuable information in such cases. This article offers a comprehensive review of conventional methods for predicting thrombus formation. In reviews conducted from the year 2000 to the present, the number of published related papers every five years has increased more than tenfold. We also found that the predictive methods can be classified into two categories: those based on the hemodynamic evaluation parameters and those based on hemodynamic and mathematical models that simulate the transport and reaction of blood components. Through our discussions, we identified several challenges that need to be resolved, including predictions based on patient-specific condition, model validation, multi-scale problems, the mechanisms of thrombus formation, and ensuring cost effectiveness. This review aims to guide researchers interested in exploring thrombus formation prediction within clinical treatments.

Finite element analysis and computational fluid dynamics to elucidate the mechanism of distal stent graft-induced new entry after frozen elephant trunk technique.

OBJECTIVES: Distal stent graft-induced new entry (dSINE), a new intimal tear at the distal edge of the frozen elephant trunk (FET), is a complication of FET. Preventive measures for dSINE have not yet been established. This study aimed to clarify the mechanisms underlying the development of dSINE by simulating the mechanical environment at the distal edge of the FET. METHODS: The stress field in the aortic wall after FET deployment was calculated using finite element analysis. Blood flow in the intraluminal space of the aorta and FET models was simulated using computational fluid dynamics. The simulations were conducted with various oversizing rates of FET ranging from 0 to 30% under the condition of FET with elastic recoil. RESULTS: The elastic recoil of the FET, which caused its distal edge to push against the greater curvature of the aorta, induced a concentration of circumferential stress and increased wall shear stress (WSS) at the aorta. Elastic recoil also created a discontinuous notch on the lesser curvature of the aorta, causing flow stagnation. An increase in the oversizing rate of the FET widened the large circumferential stress area on the greater curvature and increases the maximum stress. Conversely, a decrease in the oversizing rate of the FET increased the WSS and widened the area with high WSS. CONCLUSIONS: Circumferential stress concentration due to an oversized FET and high WSS due to an undersized FET can cause a dSINE. The selection of smaller-sized FET alone might not prevent dSINE.

Novel biaxial tensile test for studying aortic failure phenomena at a microscopic level.

BACKGROUND: An aortic aneurysm is a local dilation of the aorta, which tends to expand and often results in a fatal rupture. Although larger aneurysms have a greater risk of rupture, some small aneurysms also rupture. Since the mechanism of aortic rupture is not well understood, clarification of the microstructure influencing the failure to rupture is important. Since aortic tissues are stretched biaxially in vivo, we developed a technique to microscopically observe the failure of an aortic rupture during biaxial stretch. METHODS: A thinly sliced porcine thoracic aortic specimen was adhered to a circular frame and pushed onto a cylinder with a smaller diameter to stretch the specimen biaxially. To induce failure to rupture at the center, the specimen was thinned at the center of the hole as follows: the specimen was frozen while being compressed with metal plates having holes, which were 3 mm in diameter at their centers; the specimen was then sliced at 50-µm intervals and thawed. RESULTS: The ratio of the thickness at the center to the peripheral area was 99.5% for uncompressed specimens. The ratio decreased with an increase in the compression ratio εc and was 47.3% for specimens with εc = 40%. All specimens could be stretched until failure to rupture. The probability for crack initiation within the cylinder was <30% and 100% for specimens with εc <10% and εc >30%, respectively. Among specimens ruptured within the cylinder, 93% of those obtained from the mid-media showed crack initiation at the thin center area. CONCLUSIONS: Aortic tissues were successfully stretched biaxially until failure, and their crack initiation points were successfully observed under a microscope. This could be a very useful and powerful method for clarifying the mechanism of aortic rupture. We are planning to use this technique for a detailed investigation of events occurring at the point of failure when the crack initiates in the aortic aneurysm wall.

Delamination Strength and Elastin Interlaminar Fibers Decrease with the Development of Aortic Dissection in Model Rats.

Aortic dissection (AD) is a life-threatening tear of the vascular tissue with creation of a false lumen. To explore the mechanism underlying this tissue tear, this study investigated the delamination strength of AD model rats and the histological composition of the aorta at various stages of AD development. SD rats were administrated beta-amino propionitrile for 0 (Control), 3 (Pre-dissection), and 6 (Dissection) weeks. The thoracic aorta was harvested at 10-11 weeks of age. The Dissection group exclusively showed AD at the ascending aorta. The delamination strength, a force that separates the aorta in the radial direction, of the descending aorta decreased significantly in the order of the Control, Pre-dissection, and Dissection groups. A quantitative histological analysis of the aortic tissue demonstrated that, compared with the Control group, the area fraction of collagen was significantly higher in the Pre-dissection and Dissection groups and that of elastin was significantly lower in the Dissection group. The area fraction of the elastin fibers between the elastic laminas (interlaminar fibers) was significantly decreased in the order of the Control, Pre-dissection, and Dissection groups. Histological changes of the aortic tissue, perhaps a reduction in interlaminar fibers mainly aligned in the radial direction, decreased delamination strength, thereby causing AD.

Aneurysm Rupture Prediction Based on Strain Energy-CFD Modelling.

This paper presents a Patient-Specific Aneurysm Model (PSAM) analyzed using Computational Fluid Dynamics (CFD). The PSAM combines the energy strain function and stress-strain relationship of the dilated vessel wall to predict the rupture of aneurysms. This predictive model is developed by analyzing ultrasound images acquired with a 6-9 MHz Doppler transducer, which provides real-time data on the arterial deformations. The patient-specific cyclic loading on the PSAM is extrapolated from the strain energy function developed using historical stress-strain relationships. Multivariant factors are proposed to locate points of arterial weakening that precede rupture. Biaxial tensile tests are used to calculate the material properties of the artery wall, enabling the observation of the time-dependent material response in wall rupture formation. In this way, correlations between the wall deformation and tissue failure mode can predict the aneurysm's propensity to rupture. This method can be embedded within the ultrasound measures used to diagnose potential AAA ruptures.

Direct visualization of interstitial flow distribution in aortic walls.

Vascular smooth muscle cells are exposed to interstitial flow across aortic walls. Fluid shear stress changes the phenotype of smooth muscle cells to the synthetic type; hence, the fast interstitial flow might be related to aortic diseases. In this study, we propose a novel method to directly measure the interstitial flow velocity from the spatiotemporal changes in the concentration of a fluorescent dye. The lumen of a mouse thoracic aorta was filled with a fluorescent dye and pressurized in ex vivo. The flow of the fluorescent dye from the intimal to the adventitial sides was successfully visualized under a two-photon microscope. The flow velocity was determined by applying a one-dimensional advection-diffusion equation to the kymograph obtained from a series of fluorescent images. The results confirmed a higher interstitial flow velocity in the aortic walls under higher intraluminal pressure. A comparison of the interstitial flow velocity in the radial direction showed faster flow on the more intimal side, where hyperplasia is often found in hypertension. These results indicate that the proposed method can be used to visualize the interstitial flow directly and thus, determine the local interstitial flow velocity.

A novel method for measuring tension generated in stress fibers by applying external forces.

The distribution of contractile forces generated in cytoskeletal stress fibers (SFs) contributes to cellular dynamic functions such as migration and mechanotransduction. Here we describe a novel (to our knowledge) method for measuring local tensions in SFs based on the following procedure: 1), known forces of different magnitudes are applied to an SF in the direction perpendicular to its longitudinal axis; 2), force balance equations are used to calculate the resulting tensions in the SF from changes in the SF angle; and 3), the relationship between tension and applied force thus established is extrapolated to an applied force of zero to determine the preexisting tension in the SF. In this study, we measured tensions in SFs by attaching magnetic particles to them and applying known forces with an electromagnetic needle. Fluorescence microscopy was used to capture images of SFs fluorescently labeled with myosin II antibodies, and analysis of these images allowed the tension in the SFs to be measured. The average tension measured in this study was comparable to previous reports, which indicates that this method may become a powerful tool for elucidating the mechanisms by which cytoskeletal tensions affect cellular functions.

Stress fibers of the aortic smooth muscle cells in tissues do not align with the principal strain direction during intraluminal pressurization.

Stress fibers (SFs) in cells transmit external forces to cell nuclei, altering the DNA structure, gene expression, and cell activity. To determine whether SFs are involved in mechanosignal transduction upon intraluminal pressure, this study investigated the SF direction in smooth muscle cells (SMCs) in aortic tissue and strain in the SF direction. Aortic tissues were fixed under physiological pressure of 120 mmHg. First, we observed fluorescently labeled SFs using two-photon microscopy. It was revealed that SFs in the same smooth muscle layers were aligned in almost the same direction, and the absolute value of the alignment angle from the circumferential direction was 16.8° ± 5.2° (n = 96, mean ± SD). Second, we quantified the strain field in the aortic tissue in reference to photo-bleached markers. It was found in the radial-circumferential plane that the largest strain direction was - 21.3° ± 11.1°, and the zero normal strain direction was 28.1° ± 10.2°. Thus, the SFs in aortic SMCs were not in line with neither the largest strain direction nor the zero strain direction, although their orientation was relatively close to the zero strain direction. These results suggest that SFs in aortic SMCs undergo stretch, but not maximal and transmit the force to nuclei under intraluminal pressure.

B16 Melanoma Cancer Cells with Higher Metastatic Potential are More Deformable at a Whole-Cell Level.

INTRODUCTION: Metastasis is a process in which cancer cells spread from the primary focus site to various other organ sites. Many studies have suggested that reduced stiffness would facilitate passing through extracellular matrix when cancer cells instigate a metastatic process. Here we investigated the compressive properties of melanoma cancer cells with different metastatic potentials at the whole-cell level. Differences in their compressive properties were analyzed by examining actin filament structure and actin-related gene expression. METHODS: Compressive tests were carried out for two metastatic B16 melanoma variants (B16-F1 and B16-F10) to characterize global compressive properties of cancer cells. RNA-seq analysis and fluorescence microscopic imaging were performed to clarify contribution of actin filaments to the global compressive properties. RESULTS: RNA-seq analysis and fluorescence microscopic imaging revealed the undeveloped structure of actin filaments in B16-F10 cells. The Young's modulus of B16-F10 cells was significantly lower than that of B16-F1 cells. Disruption of the actin filaments in B16-F1 cells reduced the Young's modulus to the same level as that of B16-F10 cells, while the Young's modulus in B16-F10 cells remained the same regardless of the disruption. CONCLUSIONS: In B16 melanoma cancer cell lines, cells with higher metastatic potential were more deformable at the whole-cell level with undeveloped actin filament structure, even when highly deformed. These results imply that invasive cancer cells may gain the ability to inhibit actin filament development. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at (10.1007/s12195-021-00677-w).

Second harmonic generation light quantifies the ratio of type III to total (I + III) collagen in a bundle of collagen fiber.

The ratio of type III to type I collagen is important for properly maintaining functions of organs and cells. We propose a method to quantify the ratio of type III to total (type I + III) collagen (λIII) in a given collagen fiber bundle using second harmonic generation (SHG) light. First, the relationship between SHG light intensity and the λIII of collagen gels was examined, and the slope (k1) and SHG light intensity at 0% type III collagen (k2) were determined. Second, the SHG light intensity of a 100% type I collagen fiber bundle and its diameter (D) were measured, and the slope (k3) of the relationship was determined. The λIII in a collagen fiber bundle was estimated from these constants (k1-3) and SHG light intensity. We applied this method to collagen fiber bundles isolated from the media and adventitia of porcine thoracic aortas, and obtained λIII = 84.7% ± 13.8% and λIII = 17.5% ± 15.2%, respectively. These values concurred with those obtained with a typical quantification method using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The findings demonstrated that the method proposed is useful to quantify the ratio of type III to total collagen in a collagen fiber bundle.

Size sorting of kinesin-driven microtubules with topographical grooves on a chip.

Gliding microtubules (MTs) on a surface coated with kinesin biomolecular motors have been suggested for the development of nanoscale transport systems. In order to establish a sorting function for gliding MTs, events for MTs approaching micro-scale grooves were investigated. MTs longer than the width of grooves fabricated on a Si substrate bridged the grooves (bridging) and many MTs shorter than the groove width almost began to bridge, but returned to the surface that they approached from (guiding). Occurrence probabilities for the events were analyzed with focus on the geometric conditions, such as length of the MTs, width of the grooves, and the incident angle (alpha) of the MTs approaching the grooves. The occurrence probability for bridging increased with an increase in the incident angle (16%, alpha = 0-30 degrees; 51%, alpha = 30-60 degrees; 75%, alpha = 60-90 degrees), and the probability for guiding decreased with an increase in the incident angle (79%, alpha = 0-30 degrees; 55%, alpha = 30-60 degrees; 5%, alpha = 60-90 degrees). The results indicate that an incident angle of 30-60 degrees is an effective condition for MT sorting, because the bridging and guiding events can sort MTs that are longer and shorter than the groove widths, respectively. Furthermore, the occurrence probabilities of both bridging and guiding in a higher concentration of methylcellulose (0.5%) increased up to approximately 70% at incident angles of 30-60 degrees, indicating good feasibility for the development of devices for the sorting of MTs on surfaces with topographical grooves.

Three-dimensional analysis of the thoracic aorta microscopic deformation during intraluminal pressurization.

The aorta is composed of various constituents with different mechanical properties. This heterogeneous structure implies non-uniform deformation in the aorta, which could affect local cell functions. The present study investigates 3D strains of the aorta at a cell scale induced by intraluminal pressurization. After resected mouse, thoracic aortas were stretched to their in vivo length, and the aortas were pressurized at 15, 40, 80, 120, and 160 mmHg. Images of autofluorescent light of elastin were captured under a two-photon microscope. From the movement of markers in elastic laminas (ELs) created by photo-bleaching, 3D strains (εθθ, εzz, εrr, εrθ, εrz, εθz) between two neighboring ELs in the circumferential (θ), longitudinal (z), and radial (r) directions with reference to the dimensions at 15 mmHg were calculated. The results demonstrated that the average of shear strain εrθ was almost 0 in a physiological pressure range (from 80 to 120 mmHg) with an absolute value |εrθ| changing approximately by 5%. This indicates that ELs experience radial-circumferential shear at the cell scale, but not at the whole tissue scale. The normal strains in the circumferential εθθ and longitudinal direction εzz were positive but that in the radial direction εrr was almost 0, which demonstrates that aortic tissue is not an incompressible material. The first principal direction in the radial-circumferential plane was 29° ± 13° from the circumferential direction. We show that the aorta is not simply stretched in the circumferential direction during pressurization and that cells in the aorta undergo complex deformations by nature.

Multinucleation of Incubated Cells and Their Morphological Differences Compared to Mononuclear Cells.

Some cells cultured in vitro have multiple nuclei. Since cultured cells are used in various fields of science, including tissue engineering, the nature of the multinucleated cells must be determined. However, multinucleated cells are not frequently observed. In this study, a method to efficiently obtain multinucleated cells was established and their morphological properties were investigated. Initially, we established conditions to quickly and easily generate multinucleated cells by seeding a Xenopus tadpole epithelium tissue-derived cell line (XTC-YF) on less and more hydrophilic dishes, and incubating the cultures with medium supplemented with or without Y-27632-a ROCK inhibitor-to reduce cell contractility. Notably, 88% of the cells cultured on a less hydrophilic dish in medium supplemented with Y-27632 became multinucleate 48 h after seeding, whereas less than 5% of cells cultured under other conditions exhibited this morphology. Some cells showed an odd number (three and five) of cell nuclei 72 h after seeding. Multinucleated cells displayed a significantly smaller nuclear area, larger cell area, and smaller nuclear circularity. As changes in the morphology of the cells correlated with their functions, the proposed method would help researchers understand the functions of multinucleated cells.

Mechanophenotyping of B16 Melanoma Cell Variants for the Assessment of the Efficacy of (-)-Epigallocatechin Gallate Treatment Using a Tapered Microfluidic Device.

Metastatic cancer cells are known to have a smaller cell stiffness than healthy cells because the small stiffness is beneficial for passing through the extracellular matrix when the cancer cells instigate a metastatic process. Here we developed a simple and handy microfluidic system to assess metastatic capacity of the cancer cells from a mechanical point of view. A tapered microchannel was devised through which a cell was compressed while passing. Two metastasis B16 melanoma variants (B16-F1 and B16-F10) were examined. The shape recovery process of the cell from a compressed state was evaluated with the Kelvin⁻Voigt model. The results demonstrated that the B16-F10 cells showed a larger time constant of shape recovery than B16-F1 cells, although no significant difference in the initial strain was observed between B16-F1 cells and B16-F10 cells. We further investigated effects of catechin on the cell deformability and found that the deformability of B16-F10 cells was significantly decreased and became equivalent to that of untreated B16-F1 cells. These results addressed the utility of the present system to handily but roughly assess the metastatic capacity of cancer cells and to investigate drug efficacy on the metastatic capacity.

Photoelasticity-based evaluation of cellular contractile force for phenotypic discrimination of vascular smooth muscle cells.

Vascular smooth muscle cells (VSMCs) have two distinct phenotypes: contractile and synthetic. The major difference between these phenotypes lies in the magnitude of the contractile force produced by the cell. Although traction force microscopy (TFM) is often used to evaluate cellular contractile force, this method requires complex preprocessing and a sufficiently compliant substrate. To evaluate the contractile force and the phenotype of living VSMCs with minimal effort and in a manner independent of the substrate stiffness, we propose a photoelasticity-based method using retardation, which is related to the difference between the first and second principal stresses and their orientation. The results demonstrate that actin filaments co-localize with areas of high retardation in cells, indicating that the retardation of VSMCs is promoted by actin filaments. The retardation of cells treated with calyculin A and Y-27632 tended to be larger and smaller, respectively, than that of control cells. Cell traction force significantly correlates with total cell retardation (r2 = 0.38). The retardation of contractile VSMCs (passage 2) was significantly higher than that of synthetic VSMCs (passage 12). These results indicate that cell retardation can be used to assess cell contractile force and, thus, determine the phenotype of VSMCs.