Model for the alignment of actin filaments in endothelial cells subjected to fluid shear stress

Cultured vascular endothelial cells undergo significant morphological changes when subjected to sustained fluid shear stress. The cells elongate and align in the direction of applied flow. Accompanying this shape change is a reorganization at the intracellular level. The cytoskeletal actin filaments reorient in the direction of the cells' long axis. How this external stimulus is transmitted to the endothelial cytoskeleton still remains unclear. In this article. we present a theoretical model accounting for the cytoskeletal reorganization under the influence of fluid shear stress. We develop a system of integro-partial-differential equations describing the dynamics of actin filaments, the actin-binding proteins, and the drift of transmembrane proteins due to the fluid shear forces applied on the plasma membrane. Numerical simulations of the equations show that under certain conditions, initially randomly oriented cytoskeletal actin filaments reorient in structures parallel to the externally applied fluid shear forces. Thus, the model suggests a mechanism by which shear forces acting on the cell membrane can be transmitted to the entire cytoskeleton via molecular interactions alone.     

Cyclic stretch-induced stress fiber dynamics - Dependence on strain rate, Rho-kinase and MLCK

Stress fiber realignment is an important adaptive response to cyclic stretch for nonmuscle cells, but the mechanism by which such reorganization occurs is not known. By analyzing stress fiber dynamics using live cell microscopy, we revealed that stress fiber reorientation perpendicular to the direction of cyclic uniaxial stretching at 1 Hz did not involve disassembly of the stress fiber distal ends located at focal adhesion sites. Instead, these distal ends were often used to assemble new stress fibers oriented progressively further away from the direction of stretch. Stress fiber disassembly and reorientation were not induced when the frequency of stretch was decreased to 0.01 Hz, however. Treatment with the Rho-kinase inhibitor Y27632 reduced stress fibers to thin fibers located in the cell periphery which bundled together to form thick fibers oriented parallel to the direction of stretching at 1 Hz. In contrast, these thin fibers remained diffuse in cells subjected to stretch at 0.01 Hz. Cyclic stretch at 1 Hz also induced actin fiber formation parallel to the direction of stretch in cells treated with the myosin light chain kinase (MLCK) inhibitor ML-7, but these fibers were located centrally rather than peripherally. These results shed new light on the mechanism by which stress fibers reorient in response to cyclic stretch in different regions of the actin cytoskeleton.     

Regulation of stretch-induced JNK activation by stress fiber orientation

Cyclic mechanical stretch associated with pulsatile blood pressure can modulate cytoskeletal remodeling and intracellular signaling in vascular endothelial cells. The aim of this study was to evaluate the role of stretch-induced actin stress fiber orientation in intracellular signaling involving the activation of c-jun N-terminal kinase (JNK) in bovine aortic endothelial cells. A stretch device was designed with the capability of applying cyclic uniaxial and equibiaxial stretches to cultured endothelial cells, as well as changing the direction of cyclic uniaxial stretch. In response to 10% cyclic equibiaxial stretch, which did not result in stress fiber orientation, JNK activation was elevated for up to 6 h. In response to 10% cyclic uniaxial stretch, JNK activity was only transiently elevated, followed by a return to basal level as the actin stress fibers became oriented perpendicular to the direction of stretch. After the stress fibers had aligned perpendicularly and the JNK activity had subsided, a 90-degree change in the direction of cyclic uniaxial stretch reactivated JNK, and this activation again subsided as stress fibers became re-oriented perpendicular to the new direction of stretch. Disrupting actin filaments with cytochalasin D blocked the stress fiber orientation in response to cyclic uniaxial stretch and it also caused the uniaxial stretch-induced JNK activation to become sustained. These results suggest that stress fiber orientation perpendicular to the direction of stretch provides a mechanism for both structural and biochemical adaptation to cyclic mechanical stretch.     

Substrate deformation determines actin cytoskeleton reorganization: A mathematical modeling and experimental study

A mathematical model has been developed to define the relationship between the actin cytoskeleton reorganization of a cell and substrate deformation acting on the cell. The model is based on the following major assumptions: (a) normal substrate strain, not the shear substrate strain, determines the actin cytoskeleton reorganization; (b) the normal substrate strain is transmitted to individual actin filaments; (c) each actin filament has a basal strain energy (BSE) when the cell adheres to the substrate without stretching; and (d) the actin filaments undergo disassembly when their strain energies are decreased to zero or increased to twice their BSEs. The resulting model predicts that the actin filaments are formed in the direction where their BSEs are minimally altered. This direction is therefore the one without normal substrate strain. The prediction was confirmed by experiments conducted on both fibroblasts and endothelial cells. The present model may be relevant for understanding better the effects of mechanical stimuli on the cells.     

Microtubule dynamics regulate cyclic stretch-induced cell alignment in human airway smooth muscle cells

Microtubules are structural components of the cytoskeleton that determine cell shape, polarity, and motility in cooperation with the actin filaments. In order to determine the role of microtubules in cell alignment, human airway smooth muscle cells were exposed to cyclic uniaxial stretch. Human airway smooth muscle cells, cultured on type I collagen-coated elastic silicone membranes, were stretched uniaxially (20% in strain, 30 cycles/min) for 2 h. The population of airway smooth muscle cells which were originally oriented randomly aligned near perpendicular to the stretch axis in a time-dependent manner. However, when the cells treated with microtubule disruptors, nocodazole and colchicine, were subjected to the same cyclic uniaxial stretch, the cells failed to align. Lack of alignment was also observed for airway smooth muscle cells treated with a microtubule stabilizer, paclitaxel. To understand the intracellular mechanisms involved, we developed a computational model in which microtubule polymerization and attachment to focal adhesions were regulated by the preexisting tensile stress, pre-stress, on actin stress fibers. We demonstrate that microtubules play a central role in cell re-orientation when cells experience cyclic uniaxial stretching. Our findings further suggest that cell alignment and cytoskeletal reorganization in response to cyclic stretch results from the ability of the microtubule-stress fiber assembly to maintain a homeostatic strain on the stress fiber at focal adhesions. The mechanism of stretch-induced alignment we uncovered is likely involved in various airway functions as well as in the pathophysiology of airway remodeling in asthma.     

Cyclic Tensile Strain Controls Cell Shape and Directs Actin Stress Fiber Formation and Focal Adhesion Alignment in Spreading Cells

The actin cytoskeleton plays a crucial role for the spreading of cells, but is also a key element for the structural integrity and internal tension in cells. In fact, adhesive cells and their actin stress fiber–adhesion system show a remarkable reorganization and adaptation when subjected to external mechanical forces. Less is known about how mechanical forces alter the spreading of cells and the development of the actin–cell-matrix adhesion apparatus. We investigated these processes in fibroblasts, exposed to uniaxial cyclic tensile strain (CTS) and demonstrate that initial cell spreading is stretch-independent while it is directed by the mechanical signals in a later phase. The total temporal spreading characteristic was not changed and cell protrusions are initially formed uniformly around the cells. Analyzing the actin network, we observed that during the first phase the cells developed a circumferential arc-like actin network, not affected by the CTS. In the following orientation phase the cells elongated perpendicular to the stretch direction. This occurred simultaneously with the de novo formation of perpendicular mainly ventral actin stress fibers and concurrent realignment of cell-matrix adhesions during their maturation. The stretch-induced perpendicular cell elongation is microtubule-independent but myosin II-dependent. In summary, a CTS-induced cell orientation of spreading cells correlates temporary with the development of the acto-myosin system as well as contact to the underlying substrate by cell-matrix adhesions.     

A kinematic model coupling stress fiber dynamics with JNK activation in response to matrix stretching

The role of the actin cytoskeleton in regulating mechanotransduction in response to external forces is complex and incompletely understood. Here, we develop a mathematical model coupling the dynamic disassembly and reassembly of actin stress fibers and associated focal adhesions to the activation of c-jun N-terminal kinase (JNK) in cells attached to deformable matrices. The model is based on the assumptions that stress fibers are pre-extended to a preferred level under static conditions and that perturbations from this preferred level destabilize the stress fibers. The subsequent reassembly of fibers upregulates the rate of JNK activation as a result of the formation of new integrin bonds within the associated focal adhesions. Numerical solutions of the model equations predict that different patterns of matrix stretch result in distinct temporal patterns in JNK activation that compare well with published experimental results. In the case of cyclic uniaxial stretching, stretch-induced JNK activation slowly subsides as stress fibers gradually reorient perpendicular to the stretch direction. In contrast, JNK activation is chronically elevated in response to cyclic equibiaxial stretch. A step change in either uniaxial or equibiaxial stretch results in a short, transient upregulation in JNK that quickly returns to the basal level as overly stretched stress fibers disassemble and are replaced by fibers assembled at the preferred level of stretch. In summary, the model describes a mechanism by which the dynamic properties of the actin cytoskeleton allow cells to adapt to applied forces through turnover and reorganization to modulate intracellular signaling.     

Cyclic stretch induces reorientation of cells in a Src family kinase- and p130Cas-dependent manner

Recognition of external mechanical signals by cells is an essential process for life. One important mechanical signal experienced by various cell types, e.g. around blood vessels, within the lung epithelia or around the intestine, is cyclic stretch. As a response, many cell types reorient their actin cytoskeleton and main cell axis almost perpendicular to the direction of stretch. Despite the vital necessity of cellular adaptation to cyclic stretch, the underlying mechanosensory signal cascades are far from being understood. Here we show an important function of Src-family kinase activity in cellular reorientation upon cyclic stretch. Deletion of all three family members, namely c-Src, Yes and Fyn (SYF), results in a strongly impaired cell reorientation of mouse embryonic fibroblasts with an only incomplete reorientation upon expression of c-Src. We further demonstrate that this reorientation phenotype of SYF-depleted cells is not caused by affected protein exchange dynamics within focal adhesions or altered cell force generation. Instead, Src-family kinases regulate the reorientation in a mechanotransduction-dependent manner, since knock-down and knock-out of p130Cas, a putative stretch sensor known to be phosphorylated by Src-family kinases, also reduce cellular reorientation upon cyclic stretch. This impaired reorientation is identical in intensity upon mutating stretch-sensitive tyrosines of p130Cas only. These statistically highly significant data pinpoint early events in a Src family kinase- and p130Cas-dependent mechanosensory/mechanotransduction pathway.     

Leukotrienes and tyrosine phosphorylation mediate stretching-induced actin cytoskeletal remodeling in endothelial cells

We studied actin cytoskeletal remodeling and the role of leukotrienes and tyrosine phosphorylation in the response of endothelial cells to different types of cyclic mechanical stretching. Human aortic endothelial cells were grown on deformable silicone membranes subjected to either cyclic one-directional (strip) stretching (10%, 0.5 Hz), or biaxial stretching. After 1 min of either type of stretching, actin cytoskeletons of the stretched cells were already disrupted. After stretching for 10 and 30 min, the percentage of the stretched cells that had disrupted actin cytoskeletons were significantly increased, compared with control cells without stretching. Also, at these two time points, biaxial stretching consistently produced higher frequencies of actin cytoskeleton disruption. At 3 h, strip stretching caused the formation of stress fiber bundles, which were oriented nearly perpendicular to the stretching direction. With biaxial stretching, however, actin cytoskeletons in many stretched cells were remodeled into three-dimensional actin structures protruding outside the substrate plane, within which cyclic stretching was applied. In both stretching conditions, actin filaments were formed in the direction without substrate deformation. Moreover, substantially inhibiting either leukotriene production with nordihydroguaiaretic acid or tyrosine phosphorylation with tyrphostin A25 did not block the actin cytoskeletal remodeling. However, inhibiting both leukotriene production and tyrosine phosphorylation completely blocked the actin cytoskeletal remodeling. Thus, the study showed that the remodeling of actin cytoskeletons of the stretched endothelial cells include rapid disruption first and then re-formation. The resulting pattern of the actin cytoskeleton after remodeling depends on the type of cyclic stretching applied, but under either type of cyclic stretching, the actin filaments are formed in the direction without substrate deformation. Finally, leukotrienes and tyrosine phosphorylation are necessary for actin cytoskeletal remodeling of the endothelial cells in response to mechanical stretching.     

Nox4-dependent activation of cofilin mediates VSMC reorientation in response to cyclic stretching

Vascular smooth muscle cells (VSMCs) are subjected to various types of mechanical forces within the vessel wall. Although it is known that VSMCs undergo cell body reorientation in response to mechanical stimulation, how this mechanical stretch is transduced within the cell into biochemical signals causing cytoskeleton reorganization remains unclear. Cofilin, a protein that controls actin dynamics, is activated by Slingshot phosphatase-dependent serine 3 dephosphorylation by redox-dependent mechanisms. Nox4 is a main source of reactive oxygen species (ROS) in the vessel wall that localizes in association with the cytoskeleton. Therefore, we hypothesize that Nox4 mediates redox-dependent activation of cofilin, which is required for cytoskeletal reorganization and cell reorientation after mechanical stimulation. In this study, we found that mechanical stretch stimulates ROS production in VSMCs and that the signaling that leads to cell reorientation requires hydrogen peroxide but not superoxide. Indeed, mechanical stretch induces cofilin activation and stretch-induced cytoskeletal reorganization, and cell reorientation is inhibited in cells where cofilin activity has been downregulated. Importantly, Nox4-deficient cells fail to activate cofilin and to undergo cell reorientation, a phenotype rescued by the expression of a constitutively active cofilin mutant. Our results demonstrate that in VSMCs mechanical stimulation activates cofilin by a Nox4-dependent mechanism and that this pathway is required for cytoskeleton reorganization and cell reorientation.     

The role of Rho family proteins in controlling the migration of crawling cells

Migration of crawling cells (amoebae and some kinds of the tissue cells) is a process related to the dynamic reorganization of actomyosin cytoskeleton. That reorganization engages actin polymerization and de-polymerization, branching of actin network and interaction of myosin II with actin filaments. All those cytoskeleton changes lead to the cell progression, contraction and shifting of the uropod and the cell adhesion. Numerous external stimuli, which activate various surface receptors and signal transduction pathways, can promote migration. Rho family proteins play an important role in the regulation of actin cytoskeleton organization. The most known members of this family are Rho, Rac and Cdc42 proteins, present in all mammalian tissue cells. These proteins control three different stages of cell migration: progression of the frontal edge, adhesion which stabilizes the frontal area, and de-adhesion and shifting of the uropod. Cdc42 and Rac control cell polarization, lamellipodium formation and expansion, organization of focal complexes. Rho protein regulates contractile activity of actomyosin cytoskeleton outside the frontal area, and thus contraction and de-adhesion of the uropod.     

Jasplakinolide reversibly disrupts actin filaments in suspension-cultured tobacco BY-2 cells

Summary. Jasplakinolide is potentially a useful pharmacological tool for the study of actin organization and dynamics in living cells, since it induces actin polymerization in vitro and, unlike phalloidin, is membrane permeative. In the present work, the effect of jasplakinolide on the actin cytoskeleton of living suspension-cultured Nicotiana tabacum ‘Bright Yellow 2’ cells was investigated. Actin filaments in the living cells were disrupted by jasplakinolide. The effect of jasplakionlide on the actin cytoskeleton was concentration and time dependent. When cells were treated with a moderate concentration (150 nM) of jasplakinolide, cortical actin filaments were disrupted preferentially, whereas actin aggregated at the perinuclear region. With concentrations higher than 400 nM and exposure times longer than 30 min, actin filaments in the cell disappeared completely. The effect of jasplakinolide on the actin cytoskeleton was reversible even at high concentration. Actin bundles appeared first in the perinuclear region within 5 min, and the cortical actin array was reestablished in 15 min, suggesting that actin filaments might be organized at this region. Received July 31, 2001 Accepted December 14, 2001     

Polarization and Movement of Keratocytes: A Multiscale Modelling Approach

Eukariotic cell motility is a complex phenomenon, in which the cytoskeleton and its major constituent, actin, play an essential role. Actin forms polymers of long, stiff filaments that are cross-linked into an anisotropic network inside a thin sheet-like cellular protrusion, the lamellipod. At the leading edge of this structure, polymerization of actin filaments creates the force that pushes out the membrane and leads to translocation of a motile cell. Dynamics of the actin network account for changes in cell shape, crawling motion and turning of the cell in response to external cues. Regulating the dynamics of the cytoskeleton, and playing a central role in signal transduction in the cell, are Cdc42, Rac and Rho (GTPases of the rho family, collectively known as the small G-proteins) and the actin nucleating complex, Arp2/3.In this paper, we use a multiscale modelling approach in a 2D model of a motile cell. We describe the mutual interactions of the small G-proteins, and their effects on capping and side-branching of actin filaments. We incorporate the pushing exerted by oriented actin filament ends on the cell edge, and a Rho-dependent contraction force. Combining these biochemical and mechanical aspects, we investigate the dynamics of a model epidermal fish keratocyte through in silico experiments. Our model gives insight into how, in response to some cue, a cell can polarize, form a leading edge, and move; concomitantly it explains how a keratocyte cell can maintain its shape and polarity, even after removal of the initial stimulus, and how it can change direction quickly in response to changes in its environment. We show that establishment of polarity stems from interactions of Cdc42, Rac and Rho, while maintenance and robustness of polarity is due to the rapid cytosolic diffusion of the inactive (GDI-bound) forms of the small G-proteins. Our model produces a cell shape that closely resembles the keratocytes and correct speeds for biologically reasonable parameter values. Movies of the simulations can be obtained from http://theory.bio.uu.nl/stan/keratocyte.     

Effect of cyclic axial stretch of rat arteries on endothelial cytoskeletal morphology and vascular reactivity

Pulsatile fluid shear stress and circumferential stretch are responsible for the axial alignment of vascular endothelial cells and their actin stress fibers in vivo. We studied the effect of cyclic alterations in axial stretch independent of flow on endothelial cytoskeletal organization in intact arteries and determined if functional alterations accompanied morphologic alterations. Rat renal arteries were axially stretched (20%, 0.5 Hz) around their in vivo lengths, for up to 4h. Actin stress fibers were examined by immunofluorescent staining. We found that cyclic axial stretching of intact vessels under normal transmural pressure in the absence of shear stress induces within a few hours realignment of endothelial actin stress fibers toward the circumferential direction. Concomitant with this morphologic alteration, the sensitivity (log(EC(50))) to the endothelium-dependent vasodilator (acetylcholine) was significantly decreased in the stretched vessels (after stretching -5.15+/-0.79 and before stretching -6.71+/-0.78, resp.), while there was no difference in sodium nitroprusside (SNP) sensitivity. There was no difference in sensitivity to both acetylcholine and SNP in time control vessels. Similar to cultured cells, endothelial cells in intact vessels subjected to cyclic stretching reorganize their actin filaments almost perpendicular to the stretching direction. Accompanying this morphological alteration is a loss of endothelium-dependent vasodilation but not of smooth muscle responsiveness.     

Regulation of Actin Dynamics in Pollen Tubes: Control of Actin Polymer Level

Actin cytoskeleton undergoes rapid reorganization in response to internal and external cues. How the dynamics of actin cytoskeleton are regulated, and how its dynamics relate to its function are fundamental questions in plant cell biology. The pollen tube is a well characterized actin-based cell morphogenesis in plants. One of the striking features of actin cytoskeleton characterized in the pollen tube is its surprisingly low level of actin polymer. This special phenomenon might relate to the function of actin cytoskeleton in pollen tubes. Understanding the molecular mechanism underlying this special phenomenon requires careful analysis of actin-binding proteins that modulate actin dynamics directly. Recent biochemical and biophysical analyses of several highly conserved plant actin-binding proteins reveal unusual and unexpected properties, which emphasizes the importance of carefully analyzing their action mechanism and cellular activity. In this review, we highlight an actin monomer sequestering protein, a barbed end capping protein and an F-actin severing and dynamizing protein in plant. We propose that these proteins function in harmony to regulate actin dynamics and maintain the low level of actin polymer in pollen tubes.     

Pollen tube growth: coping with mechanical obstacles involves the cytoskeleton

Cellular growth and movement require both the control of direction and the physical capacity to generate forces. In animal cells directional control and growth forces are generated by the polymerization of and traction between the elements of the cytoskeleton. Whether actual forces generated by the cytoskeleton play a role in plant cell growth is largely unknown as the interplay between turgor and cell wall is considered to be the predominant structural feature in plant cell morphogenesis. We investigated the mechano-structural role of the cytoskeleton in the invasive growth of pollen tubes. These cells elongate rapidly by tip growth and have the ability to penetrate the stigmatic and stylar tissues in order to drill their way to the ovule. We used agents interfering with cytoskeletal functioning, latrunculin B and oryzalin, in combination with mechanical in vitro assays. While microtubule degradation had no significant effect on the pollen tubes’ capacity to invade a mechanical obstacle, latrunculin B decreased the pollen tubes’ ability to elongate in stiffened growth medium and to penetrate an obstacle. On the other hand, the ability to maintain a certain growth direction in vitro was affected by the degradation of microtubules but not actin filaments. To find out whether both cytoskeletal elements share functions or interact we used both drugs in combination resulting in a dramatic synergistic response. Fluorescent labeling revealed that the integrity of the microtubule cytoskeleton depends on the presence of actin filaments. In contrast, actin filaments seemed independent of the configuration of microtubules.     

Effect of tensile force on the mechanical behavior of actin filaments

Actin filaments are the most abundant components of the cellular cytoskeleton, and play critical roles in various cellular functions such as migration, division and shape control. In these activities, mechanical tension causes structural changes in the double-helical structure of the actin filament, which is a key modulator of cytoskeletal reorganization. This study performed large-scale molecular dynamics (MD) and steered MD simulations to quantitatively analyze the effects of tensile force on the mechanical behavior of actin filaments. The results revealed that when a tensile force of 200pN was applied to a filament consisting of 14 actin subunits, the twist angle of the filament decreased by approximately 20°, corresponding to a rotation of approximately -2° per subunit, representing a critical structural change in actin filaments. Based on these structural changes, the variance in filament length and twist angle was found to decrease, leading to increases in extensional and torsional stiffness. Torsional stiffness increased significantly under the tensile condition, and the ratio of filament stiffness under tensile force to that under no external force increased significantly on longer temporal scales. The results obtained from this study contribute to the understanding of mechano-chemical interactions concerning actin dynamics, showing that increased tensile force in the filament prevents actin regulatory proteins from binding to the filament.     

A kinematic model of stretch-induced stress fiber turnover and reorientation

A kinetic model based on constrained mixture theory was developed to describe the reorganization of actin stress fibers in adherent cells in response to diverse patterns of mechanical stretch. The model was based on reports that stress fibers are pre-extended at a “homeostatic” level under normal, non-perturbed conditions, and that perturbations in stress fiber length destabilize stress fibers. In response to a step change in matrix stretch, the model predicts that stress fibers are initially stretched in registry with the matrix, but that these overly stretched fibers are gradually replaced by new fibers assembled with the homeostatic level of stretch in the new configuration of the matrix. In contrast, average fiber stretch is chronically perturbed from the homeostatic level when the cells are subjected to cyclic equibiaxial stretch. The model was able to describe 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. The model also accurately described the relationship between stretch magnitude and the extent of stress fiber alignment in endothelial cells subjected to cyclic uniaxial stretch. Further, in the case of cyclic simple elongation with transverse matrix contraction, stress fibers orient in the direction of least perturbation in stretch. In summary, the model predicts that the rate of stretch-induced stress fiber disassembly determines the rate of alignment, and that stress fibers tend to orient toward the direction of minimum matrix stretch where the rate of stress fiber turnover is a minimum.     

Role of Cytoskeleton Proteins in the Morphological Changes During Apoptotic Cell Death of Cerebellar Granule Neurons

Cytoskeleton proteins are substrates for proteases and further apoptotic death. We evaluated the participation of cytoskeleton in morphological changes during cell death induced by two apoptotic conditions, potassium deprivation (K5) and staurosporine, in cerebellar granule neurons (CGC). We found that K5 induced somatic damage, but neurites were relatively preserved, which corresponded to the reorganization of actin and α-tubulin in neurites. Staurosporine (STS) induced an early alteration of neurites with reorganization of cytoskeleton proteins in somas. Caspase inhibitor ZVAD totally inhibited STS-induced α-tubulin reorganization and partially blocked STS-induced actin reorganization. α-tubulin and actin reorganization induced by K5 was affected by ZVAD. Calpain inhibitor (IC1) did not affect α-tubulin or actin reorganization induced by STS, K5 or ionomycin. Neither ZVAD, nor IC1 changed α-tubulin or actin levels upon K5 treatment. STS increased α-tubulin and actin levels, but neither ZVAD nor IC1 changed α-tubulin levels upon STS treatment. In contrast, ZVAD reduced the STS-induced increase of actin. These results suggest that CGC cytoskeleton proteins undergo a differential expression and reorganization depending on the apoptotic condition.     

Stepwise morphological changes and cytoskeletal reorganization of human mesenchymal stem cells treated by short-time cyclic uniaxial stretch

This study aimed to investigate stepwise remodeling of human mesenchymal stem cells (hMSCs) in response to cyclic stretch through rearrangement and alignment of cells and cytoskeleton regulation toward smooth muscle cell (SMC) fate in different time spans. Image analysis techniques were utilized to calculate morphological parameters. Cytoskeletal reorganization was observed by investigating F-actin filaments using immunofluorescence staining, and expression level of contractile SMC markers was followed by a quantitative polymerase chain reaction method. Applying cyclic uniaxial stretch on cultured hMSCs, utilizing a costume-made device, led to alteration in fractal dimension (FD) and cytoskeleton structure toward continuous alignment and elongation of cells by elevation of strain duration. Actin filaments became more aligned perpendicular to the axis of mechanical stretch by increasing uniaxial loading duration. At first, FD met a significant decrease in 4 h loading duration then increased significantly by further loading up to 16 h, followed by another decrease up to 1 d of uniaxial stretching. HMSCs subjected to 24 h cyclic uniaxial stretching significantly expressed early and intermediate contractile SM markers. It was hypothesized that the increase in FD after 4 h while cells continuously became more aligned and elongated was due to initiation of change in phenotype that influenced arrangement of cells. At this point, change in cell phenotype started leading to change in morphology while mechanical loading still caused cell alignment and rearrangement. Results can be helpful when optimized engineered cells are needed based on mechanical condition for functional engineered tissue and cell therapy.