Unstretched Cells Research Articles

Influence of circadian rhythm on effects induced by mechanical strain in periodontal ligament cells.

The aim of this study was to investigate the influence of mechanical strain on clock gene function in periodontal ligament (PDL) cells. Furthermore, we wanted to analyze whether effects induced by mechanical stress vary in relation to the circadian rhythm. Human PDL fibroblasts were synchronized in their circadian rhythm with dexamethasone and stretched over 24 h. Unstretched cells served as controls. Gene expression of the core clock genes were analyzed at 4 h intervals by quantitative real-time polymerase chain reaction (qRT-PCR). Time points 0 h (group SI1) and 12 h (group SI2) after synchronization served as starting points of a4 h force application period. Collagen-1α (COL-1α/Col-1α), interleukin-1β (IL1-β), and runt-related transcription factor 2 (RUNX2/Runx2) were assessed by qRT-PCR and enzyme-linked immunosorbent assay (ELISA) after 2 and 4 h. Statistical analysis comprised one-way analysis of variance (ANOVA) and posthoc tests. After synchronization, the typical pattern for clock genes was visible in control cells over the 24 h period. This pattern was significantly altered by mechanical strain. Under tensile stress, ARNTL gene expression was reduced, while Per1 and2 gene expression were upregulated. In addition, mechanical stress had adifferential effect on the expression of Col-1α and IL1‑β depending on its initiation within the circadian rhythm (group SI1 vs group SI2). For RUNX2, no significant differences in the two groups were observed. Our results suggest that mechanical stress affects the molecular peripheral oscillator of PDL cells. Vice versa, the circadian rhythm also seems to partially influence the effects that mechanical stress exerts on PDL cells.

Mechanical stretch promotes apoptosis and impedes ciliogenesis of primary human airway basal stem cells

BackgroundAirway basal stem cells (ABSCs) have self-renewal and differentiation abilities. Although an abnormal mechanical environment related to chronic airway disease (CAD) can cause ABSC dysfunction, it remains unclear how mechanical stretch regulates the behavior and structure of ABSCs. Here, we explored the effect of mechanical stretch on primary human ABSCs.MethodsPrimary human ABSCs were isolated from healthy volunteers. A Flexcell FX-5000 Tension system was used to mimic the pathological airway mechanical stretch conditions of patients with CAD. ABSCs were stretched for 12, 24, or 48 h with 20% elongation. We first performed bulk RNA sequencing to identify the most predominantly changed genes and pathways. Next, apoptosis of stretched ABSCs was detected with Annexin V-FITC/PI staining and a caspase 3 activity assay. Proliferation of stretched ABSCs was assessed by measuring MKI67 mRNA expression and cell cycle dynamics. Immunofluorescence and hematoxylin-eosin staining were used to demonstrate the differentiation state of ABSCs at the air-liquid interface.ResultsCompared with unstretched control cells, apoptosis and caspase 3 activation of ABSCs stretched for 48 h were significantly increased (p < 0.0001; p < 0.0001, respectively), and MKI67 mRNA levels were decreased (p < 0.0001). In addition, a significant increase in the G0/G1 population (20.2%, p < 0.001) and a significant decrease in S-phase cells (21.1%, p < 0.0001) were observed. The ratio of Krt5+ ABSCs was significantly higher (32.38% vs. 48.71%, p = 0.0037) following stretching, while the ratio of Ac-tub+ cells was significantly lower (37.64% vs. 21.29%, p < 0.001). Moreover, compared with the control, the expression of NKX2-1 was upregulated significantly after stretching (14.06% vs. 39.51%, p < 0.0001). RNA sequencing showed 285 differentially expressed genes, among which 140 were upregulated and 145 were downregulated, revealing that DDIAS, BIRC5, TGFBI, and NKX2-1 may be involved in the function of primary human ABSCs during mechanical stretch. There was no apparent difference between stretching ABSCs for 24 and 48 h compared with the control.ConclusionsPathological stretching induces apoptosis of ABSCs, inhibits their proliferation, and disrupts cilia cell differentiation. These features may be related to abnormal regeneration and repair observed after airway epithelium injury in patients with CAD.

HMGB1/RAGE axis accelerates the repair of HUVECs injured by pathological mechanical stretching via promoting bFGF expression

AimDuring hypertension-induced endothelial dysfunction, periodic mechanical stretching (MS) activates related inflammatory pathways and leads to endothelial damage, but the underlying mechanisms remain unknown. The present study aimed to determine the injury of HUVECs caused by overstretching and the role of HMGB1-RAGE pathway in HUVECs after injury. Main methods and key findingsHuman umbilical vein endothelial cells (HUVECs) were cultured and seeded in BioFlex™ plates (six wells). Cells were exposed to 5% (physiological state) and 20% (pathological state) mechanical stretch at 1 Hz for 12 or 24 h in a Flexcell-5000™, with unstretched cells serving as controls. It was found that excessive MS can inhibit cell viability, proliferation, and tube-forming ability resulting in disordered cell arrangement and orientation, slowing cell migration. All these changes cause endothelial damage compared to physiological MS. Endothelial cells (ECs) promote cell migration and self-repair after injury by increasing the High-mobility group box 1 (HMGB1) expression. Experiments and protein prediction networks have shown that HMGB1 can also promote the expression of downstream protein bFGF by binding to receptor for advanced glycation end products (RAGE). Interestingly, VEGF protein expression did not change significantly during this repair process, implying that bFGF replaces the role of VEGF in vascular endothelial repair. SignificanceThe present study demonstrates that in the context of endothelial injury caused by excessive MS, the HMGB1/RAGE/bFGF pathway is activated and promotes endothelial repair after injury. Therefore, understanding these mechanisms will help find new therapies for diseases such as hypertension, atherosclerosis, and aneurysm formation.

Differential Expression of Adipokines in Mechanically Stretched‐ Endothelial cells

IntroductionHypertension is considered as the major risk factor for cardiovascular diseases, which are the leading cause of morbidity and mortality worldwide. This disease can develop mainly due to the mechanical forces exerted by the blood on the vessels wall. The endothelium, lining the blood vessel, responds to these hemodynamic mechanical forces such as mechanical stretch leading to endothelial dysfunction. Leptin and adiponectin (APN) are two adipokines that are known to be associated with the pathogenesis of hypertension. However, how the level of these hormones is elevated in response to mechanical stretch in endothelial cells is yet to be elucidated.AimIn the present study, we investigated the expression of leptin and adiponectin in endothelial cells exposed to mechanical stretch.MethodsImmortalized human umbilical vein endothelial cells (EA. hy926) were subjected to mechanical stretch (15%, 60 cycle/min and 1Hz) using STREX cell stretching system (STREX Inc., California, USA) for 6, 16 and 24 h. Cultured cells not subjected to stretch (static condition) where used as controls. The mRNA expression of leptin and its receptor (OB‐Rb) as well as APN and adiponectin receptor 2 (APN‐R2) were assessed at different time points using real‐time PCR.ResultsMechanical stretch induced changes in cell morphology in which the cells were elongated and aligned perpendicular to the stretch axis. Stretching of endothelial cells significantly upregulated the expression of leptin after 16 h (0.9515 ± 0.4238‐fold, n=6, p&lt;0.05) and after 24 h (1.577 ± 0.4503‐fold, n=6, p&lt;0.05) of stretching but without affecting the expression of its receptor (OB‐Rb). The mRNA expression of APN also increased significantly in response to 6 h stretching (0.5457 ± 0.1890‐fold, n=6, p&lt;0.05), 16 h stretching (0.9487 ± 0.4058‐fold, n=6, p&lt;0.05) and 24 h stretching (2.664 ± 0.3447, n=6, p&lt;0.0001) compared to unstretched cells, however no change in the expression of APN‐R2 was observed.ConclusionOur findings suggesting that the exposure of endothelial cells to mechanical stretch can result in changes in the expression of adipokines, leptin and adiponectin, will result in better understanding of how vascular biology is affected by mechanical forces and how these molecules can modulate endothelial function in cardiovascular diseases.

Cyclically stretched ACL fibroblasts emigrating from spheroids adapt their cytoskeleton and ligament-related expression profile

Mechanical stress of ligaments varies; hence, ligament fibroblasts must adapt their expression profile to novel mechanomilieus to ensure tissue resilience. Activation of the mechanoreceptors leads to a specific signal transduction, the so-called mechanotransduction. However, with regard to their natural three-dimensional (3D) microenvironment cell reaction to mechanical stimuli during emigrating from a 3D spheroid culture is still unclear. This study aims to provide a deeper understanding of the reaction profile of anterior cruciate ligament (ACL)-derived fibroblasts exposed to cyclic uniaxial strain in two-dimensional (2D) monolayer culture and during emigration from 3D spheroids with respect to cell survival, cell and cytoskeletal orientation, distribution, and expression profile. Monolayers and spheroids were cultured in crosslinked polydimethyl siloxane (PDMS) elastomeric chambers and uniaxially stretched (14% at 0.3 Hz) for 48 h. Cell vitality, their distribution, nuclear shape, stress fiber orientation, focal adhesions, proliferation, expression of ECM components such as sulfated glycosaminoglycans, collagen type I, decorin, tenascin C and cell–cell communication-related gap junctional connexin (CXN) 43, tendon-related markers Mohawk and tenomodulin (myodulin) were analyzed. In contrast to unstretched cells, stretched fibroblasts showed elongation of stress fibers, cell and cytoskeletal alignment perpendicular to strain direction, less rounded cell nuclei, increased numbers of focal adhesions, proliferation, amplified CXN43, and main ECM component expression in both cultures. The applied cyclic stretch protocol evoked an anabolic response and enhanced tendon-related marker expression in ACL-derived fibroblasts emigrating from 3D spheroids and seems also promising to support in future tissue formation in ACL scaffolds seeded in vitro with spheroids.

Stretch synchronizes sarcomere shortening in isolated cardiomyocytes

According to the Frank-Starling mechanism, stretching cardiac muscle augments contractile force. However, it is unclear how this phenomenon occurs regionally within cardiomyocytes, at the level of individual sarcomeres and their local Ca2+ signals. To investigate this issue, we simultaneously monitored cytosolic Ca2+ and sarcomere striations in isolated rat cardiomyocytes by recording Fluo-4 fluorescence (515–560 nm) and brightfield signals respectively, using a high-speed sCMOS camera. Cells were affixed to glass rods (CellTester, World Precision Instruments) to allow control of cell length and measurement of force production during electrical stimulation at 1 Hz. At resting sarcomere length (SL), differential sarcomere deformation was observed despite uniform Ca2+ transients corresponding to individual sarcomeres. Specifically, while most sarcomeres contracted during the stimulus, a minority of sarcomeres (~13%) were observed to be stretched or were stationary. Sarcomeres which exhibited stretch during the Ca2+ transient tended to be shorter than those that contracted, and were significantly shorter than their neighbours. Step-wise stretch of the cell generated an expected increase in force development, but also revealed that more sarcomeres exhibited contraction, reaching nearly 100% of sarcomeres at 20% stretch. During cell lengthening, resting SL increased in all sarcomeres, suggesting that the shortest sarcomeres surpassed a critical length which enabled sufficient force generation and shortening. SL variability in unstretched cells was not caused by regional differences in Ca2+, as it remained apparent during permeabilization in Ca2+-free conditions. Rather, electron microscopy analysis traced differences in SL to the I-band, where dimensions are regulated by titin. We postulate, therefore, that slackened titin in unstretched cells allows greater variability in I-band length, cross-bridge formation, and active force generation between sarcomeres. When the cell is lengthened, sarcomere shortening is synchronized, which may contribute to augmented force production defined by the Frank-Starling mechanism.

Roles of TRPV4 and piezo channels in stretch-evoked Ca2+ response in chondrocytes.

Chondrocyte mechanotransduction is not well understood, but recently, it has been proposed that mechanically activated ion channels such as transient receptor potential vanilloid 4 (TRPV4), Piezo1, and Piezo2 are of functional importance in chondrocyte mechanotransduction. The aim of this study was to distinguish the potential contributions of TRPV4, Piezo1, and Piezo2 in transducing different intensities of repetitive mechanical stimulus in chondrocytes. To study this, TRPV4-, Piezo1-, or Piezo2-specific siRNAs were transfected into cultured primary chondrocytes to knock down (KD) TRPV4, Piezo1, or Piezo2 expression, designated TRPV4-KD, Piezo1-KD, or Piezo2-KD cells. Then we used Flexcell® Tension System to apply cyclic tensile strains (CTS) of 3% to 18% at 0.5 Hz for 8 h to the knockdown and control siRNA-treated cells. Finally, using a Ca2+ imaging system, stretch-evoked intracellular Ca2+ ([Ca2+] i) influx in chondrocytes was examined to investigate the roles of TRPV4, Piezo1, and Piezo2 in Ca2+ signaling in response to different intensities of repetitive mechanical stretch stimulation. The characteristics of [Ca2+] i in chondrocytes evoked by stretch stimulation were stretch intensity dependent when comparing unstretched cells. In addition, stretch-evoked [Ca2+] i changes were significantly suppressed in TRPV4-KD, Piezo1-KD, or Piezo2-KD cells compared with control siRNA-treated cells, indicating that any channel essential for Ca2+ signaling induced by stretch stimulation in chondrocytes. Of note, they played different roles in calcium oscillation induced by different intensities of stretch stimulation. More specifically, TRPV4-mediated Ca2+ signaling played a central role in the response of chondrocytes to physiologic levels of strain (3% and 8% of strain), while Piezo2-mediated Ca2+ signaling played a central role in the response of chondrocytes to injurious levels of strain (18% of strain). These results provide a basis for further examination of mechanotransduction in cartilage and raise a possibility of therapeutically targeting Piezo2-mediated mechanotransduction for the treatment of cartilage disease induced by repetitive mechanical forces. Impact statement Chondrocytes in cartilage are constantly subjected to load-induced stimuli and regulate their metabolic activities in order to maintain cartilage homeostasis. Therefore, mechanotransduction is important in chondrocytes and is vital for their role in cartilage function. Our results indicate that chondrocytes might sense and distinguish the different intensities of repetitive mechanical stimulus by using different mechanosensitive ion channels. Specifically, TRPV4 is mainly responsible for sensing physiologic levels of repetitive CTS stimulus, while Piezo2 mainly contributes to chondrocyte sensing noxious levels of repetitive CTS loading. These results provide a basis for further examination of mechanotransduction in cartilage and raise the possibility of therapeutically targeting Piezo2-mediated mechanotransduction for the treatment of OA which is induced by injurious and repetitive mechanical stimulation.

Uniaxial Cyclic Tensile Stretching at 8% Strain Exclusively Promotes Tenogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stromal Cells.

The present study was conducted to establish the amount of mechanical strain (uniaxial cyclic stretching) required to provide optimal tenogenic differentiation expression in human mesenchymal stromal cells (hMSCs) in vitro, in view of its potential application for tendon maintenance and regeneration. Methods. In the present study, hMSCs were subjected to 1 Hz uniaxial cyclic stretching for 6, 24, 48, and 72 hours; and were compared to unstretched cells. Changes in cell morphology were observed under light and atomic force microscopy. The tenogenic, osteogenic, adipogenic, and chondrogenic differentiation potential of hMSCs were evaluated using biochemical assays, extracellular matrix expressions, and selected mesenchyme gene expression markers; and were compared to primary tenocytes. Results. Cells subjected to loading displayed cytoskeletal coarsening, longer actin stress fiber, and higher cell stiffness as early as 6 hours. At 8% and 12% strains, an increase in collagen I, collagen III, fibronectin, and N-cadherin production was observed. Tenogenic gene expressions were highly expressed (p < 0.05) at 8% (highest) and 12%, both comparable to tenocytes. In contrast, the osteoblastic, chondrogenic, and adipogenic marker genes appeared to be downregulated. Conclusion. Our study suggests that mechanical loading at 8% strain and 1 Hz provides exclusive tenogenic differentiation; and produced comparable protein and gene expression to primary tenocytes.

The Effects of Mechanical Load on Transmural Differences in Mechano-Electric Feedback in Single Cardiomyocytes

We have shown previously that subendocardial (ENDO) and subepicardial (EPI) cardiomyocytes isolated from the mouse left ventricle (LV) have different mechanical responses to changes in preload (stretch) to the cells (Khokhlova et al., 2018). However, the main limitation of previous study was small amount of stretch (about 3.5%) applied. Here we resolved the limitation applying up to 20% of stretch and used our new electromechanical ENDO and EPI mathematical models to predict effects of mechanical load on electrophysiological function of ENDO and EPI cells. Various preloads imposed via axial stretch were applied to the mouse LV cells using carbon fiber technique for single myocytes (Iribe et al., 2014). To simulate experimentally obtained results and to predict mechanisms underlying the cellular response to change in preload we developed mathematical models of the mouse ENDO and EPI cells. In consistency with the previous results, under auxotonic contractions at low preload (unstretched cells), time to peak contraction (Tmax) was significantly longer in ENDO cells than in EPI cells. An increase in preload (stretch) prolonged Tmax in both cell types, however, the slope of “Tmax - sarcomere stretch” relationship was significantly higher in EPI cells, demonstrating greater contraction sensitivity of EPI cells to the stretch. Our mathematical cellular models reproduced the experimental results when we accounted for the ENDO/EPI differences in the parameters of cooperativity of calcium activation of myofilaments. The models predicted greater prolongation of action potential in EPI cells under high preload. Our results suggest that mechanical load changes transmural gradient in contraction of ENDO and EPI cardiomyocytes that in turn affect the gradient in electrophysiological function via mechano-calcium-electric feedback. Supported by The Russian Science Foundation (#18-74-10059) and JSPS KAKENHI (#16K12878).

The effects of load on transmural differences in contraction of isolated mouse ventricular cardiomyocytes

Mechanical properties of cardiomyocytes from different transmural regions are heterogeneous in the left ventricular wall. The cardiomyocyte mechanical environment affects this heterogeneity because of mechano-electric feedback mechanisms. In the present study, we investigated the effects of the mechanical load (preload and afterload) on transmural differences in contraction of subendocardial (ENDO) and subepicardial (EPI) single cells isolated from the murine left ventricle. Various preloads imposed via axial stretch and afterloads (unloaded and heavy loaded conditions) were applied to the cells using carbon fiber techniques for single myocytes. To simulate experimentally obtained results and to predict mechanisms underlying the cellular response to change in load, our mathematical models of the ENDO and EPI cells were used.Our major findings are the following. Our results show that ENDO and EPI cardiomyocytes have different mechanical responses to changes in preload to the cells. Under auxotonic contractions at low preload (unstretched cells), time to peak contraction (Tmax) and the time constant of [Ca2+]i transient decay were significantly longer in ENDO cells than in EPI cells. An increase in preload (stretched cells) prolonged Tmax in both cell types; however, the prolongation was greater in EPI cells, resulting in a decrease in the transmural gradient in Tmax at high preload. Comparing unloaded and heavy loaded (isometric) contractions of the cells we found that transmural gradient in the time course of contraction is independent of the loading conditions. Our mathematical cell models were able to reproduce the experimental results on the distinct cellular responses to changes in the mechanical load when we accounted for an ENDO/EPI difference in the parameters of cooperativity of calcium activation of myofilaments.

Induction of MMP-1 and −3 by cyclical mechanical stretch is mediated by IL-6 in cultured fibroblasts of keratoconus

In order to understand the effect of mechanical stretch on corneal extracellular matrix remodeling, human keratoconus fibroblasts (HKCFBs) were subjected to cyclic stretch invitro and the expression of matrix metalloproteinases (MMPs), tissue inhibitor of metalloproteinases (TIMPs), and inflammatory cytokines were evaluated. HKCFBs were seeded into a flexible membrane base and subjected to a cyclic stretch regimen of 10% equibiaxial stretch at a stretching frequency of 1Hz for 6h using a Flexcell tension unit. An antibody directed against interleukin‑6 (IL‑6 Ab) was used to investigate the roles of IL‑6 on mechanical stretch mediated regulation of MMP in HKCFBs. Culture supernatants were assayed using an enzyme‑linked immunosorbent assay for MMP‑1 and ‑3, TIMP‑1 and ‑2, and IL‑6. Total RNA from the cells was extracted, and quantitative polymerase chain reaction was used to determine mRNA for MMP‑1 and ‑3, TIMP‑1 and ‑2, and IL‑6. In stretched cells, levels of MMP‑1 and ‑3 demonstrated an increase compared with unstretched cells, but levels of TIMP‑1, and ‑2 revealed a decrease. Mechanical stretch significantly increased the mRNA expression and protein synthesis of IL‑6 compared with unstretched cells. IL‑6 induced MMP‑1 and ‑3 expression, whereas no significant effects were observed in levels of TIMP‑1 and ‑2 compared with the untreated control groups. Additionally, the IL‑6 Ab markedly inhibited the stretch‑induced increase in MMP‑1 and ‑3 in culture supernatants in a dose‑dependent manner. No significant differences in TIMP‑1 and ‑2 protein levels were detected between stretched cells treated with IL‑6 Ab and stretched cells without IL‑6 Ab treatment. These results indicate that cyclical mechanical stretch augments IL‑6 production and MMP expression, and reduces levels of TIMP in HKCFBs. Thus, it is suggested that IL‑6 mediates the stretch‑induced MMP expression.

Effect of cyclic stretching on cell shape and division

In multicellular organisms, cell fate and tissue organization are greatly affected by the orientation of the cell division axis. Cell division is aligned perpendicular to the long axis of a cell - the so-called ‘Hertwig’s rule’. Because mechanical strains on cells cause arrangement of cytoskeleton molecules which determines cell shape, it is expected that mechanical strains also play an important role on the orientation of the cell division axis. In this study, we used a microfabricated cyclic stretching device to understand how mechanical strains affect the orientation of the cell division axis, which determines the spatial arrangement of daughter cells. When RPE-1 (retinal pigmented epithelial) cells were cyclically stretched during their prophase and prometa phase at various stretching magnitudes (5% and 10%) and frequencies (0.1-10 Hz), the cells’ aspect ratios were at their highest. Compared to unstretched cells with aspect ratios below 2.5, the stretched ones with aspect ratios higher than 4 better suited Hertwig’s rule by showing a good alignment of spindle angle to the perpendicular direction to the long axis of their cell bodies. These results suggest that mechanical strains play important roles in determining the cell division axis by affecting cell shape and orientation.

Expression of heat shock protein 27 correlates with actin cytoskeletal dynamics and contractility of cultured human bladder smooth muscle cells

Heat shock protein 27 (HSP27) is one of the most important chaperone proteins that modulates smooth muscle contraction. Here we investigated the effects of HSP27 expression on cytoskeleton dynamics and contractile function of human bladder smooth muscle cells (BSMCs) in vitro. Cultured human BSMCs were transfected with lentiviral vectors expressing either HSP27 or HSP27-siRNAs. Normal BSMCs cells and cells transfected with the empty lentivirus were used as control. Cells were then cultured on Flexcell flexible membrane dishes and mechanical stretch (14.8% elongation) was applied. The stretch caused significant disruption of actin cytoskeletal structure and decrease in F/G-actin ratio in BSMCs with HSP27 over-expression, knock-down and control groups (P<0.05) as indicated by phalloidin-FITC staining. It was also shown that the structure of actin filaments in HSP27 over-expressed cells recovered and F/G-actin ratio significantly increased at 12h after stretching compared to unstretched cells (P<0.05), but not in HSP27 knock-down cells, suggesting that HSP27 promoted the recovery of cytoskeletal structure in BSMCs from stretch-induced injury. In addition, the contractile force of BSMCs was enhanced by over-expression of HSP27 and attenuated by knock-down of HSP27 (P<0.05), suggesting a pivotal role of HSP27 in regulating bladder smooth muscle contraction.

Transplantation of Cyclic Stretched Fibroblasts Accelerates the Wound-Healing Process in Streptozotocin-Induced Diabetic Mice

Mechanical stimulation is a known modulator of survival and proliferation for many cells, including endothelial cells, smooth muscle cells, and bone marrow-derived mesenchymal stem cells. In this study, we found that mechanical strain prevents apoptosis and increases the adhesive ability of dermal fibroblasts in vitro and thus confers the survival advantage in vivo after transplantation of fibroblasts into the full-thickness wound of diabetic mice. Cyclic stretch at a frequency of 0.5 Hz and maximum elongation of 20% stimulates cellular survival mediated by the activation of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and the serine/threonine kinase Akt (AKT). Stretching of the fibroblasts increases the synthesis of extracellular matrix proteins and the formation of denser focal adhesion structures, both of which are required for fibroblast adhesion. The stretched fibroblasts also upregulate the expression of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α), which enhanced wound healing in vivo. Indeed, preconditioning with mechanical stretch allows better survival of the transplanted fibroblasts, when compared to unstretched control cells, in the wound environment of mice with streptozotocin-induced diabetes and thus accelerates the wound-healing process in these mice.

Biological effects of cellular stretch on human dermal fibroblasts

Pathological scars are fibroproliferative skin disorders that are characterised by the accumulation of fibroblasts and collagens. It is increasingly understood that their development and progression may be related to local skin mechanics, such as stretching. The present study evaluated the morphological and functional effects of cellular stretch on normal human dermal fibroblasts and explored the mechanotransduction mechanisms that may be involved. When fibroblasts were subjected to 24h of cyclic axial stretching (10cyclesmin(-1)), they migrated faster and for a longer distance than unstretched cells. The increased migration resulted in the cells reorienting themselves perpendicular to the direction of stretching. This was associated with reduced cellular apoptosis and unchanged proliferation. Stretching did not increase collagen synthesis but did elevate collagen degradation. These biological effects appeared to be mediated by the integrin and Wnt mechanotransduction pathways, which transmitted the mechanical stimulus via cell-substrate interactions, cell-cell junctions and indirect cell-cell communications. A better understanding of such fibroblast mechanoresponses invitro will help the development of novel interventions that can prevent, reduce or even reverse pathological scar formation and/or progression invivo.

Cyclic Stretch–Induced Oxidative Stress Increases Pulmonary Alveolar Epithelial Permeability

Mechanical ventilation with high tidal volumes has been associated with pulmonary alveolar flooding. Understanding the mechanisms underlying cyclic stretch-induced increases in alveolar epithelial permeability may be important in designing preventive measures for acute lung injury. In this work, we assessed whether cyclic stretch leads to the generation of reactive oxygen species in type I-like alveolar epithelial cells, which increase monolayer permeability via activation of NF-κB and extracellular signal-regulated kinase (ERK). We cyclically stretched type I-like rat primary alveolar epithelial cells at magnitudes of 12, 25, and 37% change in surface area (ΔSA) for 10 to 120 minutes. High levels of reactive oxygen species and of superoxide and NO specifically were detected in cells stretched at 37% ΔSA for 10 to 120 minutes. Exogenous superoxide and NO stimulation increased epithelial permeability in unstretched cells, which was preventable by the NF-κB inhibitor MG132. The cyclic stretch-induced increase in permeability was decreased by the superoxide scavenger tiron and by MG132. Furthermore, tiron had a dramatic protective effect on in vivo lung permeability under mechanical ventilation conditions. Cyclic stretch increased the activation of the NF-κB signaling pathway, which was significantly decreased with the ERK inhibitor U0126. Altogether, our in vitro and in vivo data demonstrate the sensitivity of permeability to stretch- and ventilation-induced superoxide production, suggesting that using antioxidants may be helpful in the prevention and treatment of ventilator-induced lung injury.

Rac1 pathway mediates stretch response in pulmonary alveolar epithelial cells

Alveolar epithelial cells (AECs) maintain the pulmonary blood-gas barrier integrity with gasketlike intercellular tight junctions (TJ) that are anchored internally to the actin cytoskeleton. We have previously shown that AEC monolayers stretched cyclically and equibiaxially undergo rapid magnitude- and frequency-dependent actin cytoskeletal remodeling to form perijunctional actin rings (PJARs). In this work, we show that even 10 min of stretch induced increases in the phosphorylation of Akt and LIM kinase (LIMK) and decreases in cofilin phosphorylation, suggesting that the Rac1/Akt pathway is involved in these stretch-mediated changes. We confirmed that Rac1 inhibitors wortmannin or EHT-1864 decrease stretch-stimulated Akt and LIMK phosphorylation and that Rac1 agonists PIP3 or PDGF increase phosphorylation of these proteins in unstretched cells. We also confirmed that Rac1 pathway inhibition during stretch modulated stretch-induced changes in occludin content and monolayer permeability, actin remodeling and PJAR formation, and cell death. As further validation, overexpression of Rac GTPase-activating protein β2-chimerin also preserved monolayer barrier properties in stretched monolayers. In summary, our data suggest that constitutive activity of Rac1, which is necessary for stretch-induced activation of the Rac1 downstream proteins, mediates stretch-induced increases in permeability and PJAR formation.

Stretch-Induced Activation of NFAT Signaling in Hl-1 Cardiomyocytes

Mechanical forces are efficiently converted into cellular signals to initiate structural and functional remodeling of cardiac myocytes. Molecular mechanisms underlying cardiac mechanotransduction are still barely understood. Nuclear Factor of Activated T-cells (NFAT) is an important calcium-responsive transcription factor and a key player in cardiac remodeling processes. In this study, we investigated the involvement of Ca2+ signaling molecules as a link between mechanical stretch and NFAT translocation in HL-1 atrial myocytes. We specifically focused on the potential role of receptor-operated and/or store-operated Ca2+ permeable channels of the TRPCs (classical transient receptor potential) and the Orai family. Populations of HL-1 cells overexpressing GFP-NFAT were exposed to a single-stretch by 20% for 20 minutes on commercially available silicon membranes and NFAT localization was then analyzed by fluorescence microscopy. Results revealed that stretched HL-1 cells significantly increased NFAT nuclear translocation when compared to unstretched cells. Similar NFAT activation was obtained when HL-1 cells were stimulated by either endothelin-1 (100 nM) or thapsigargin (1μM) in the absence of a stretch stimulus. These results demonstrate that similar levels of NFAT activation are induced in HL-1 cells by either mechanical stress, cell activation via Gq-coupled receptors or depletion of internal Ca2+ stores. HL-1 cells were found to express several TRPC isoforms that might contribute to mechanotransduction. Our data suggest a linkage between mechanical stretch and cardiac remodeling involving Ca2+ entry and NFAT activation.This work is funded by FWF (Austrian research fund) projects P21925-B19, P22565-B18 and the DK Metabolic and Cardiovascular Disease grant W1226-B18.

Mechanoregulation of osteoblast-like MG-63 cell activities by cyclic stretching

Mechanical loading plays an important role in regulating bone formation and remodeling. Relevant mechanical stretching can increase the proliferation and differentiation of osteoblastic cells invitro. However, little is known about the effects of supraphysiological high-level mechanical stretching on the growth and cell cycle progression of osteoblastic cells. Osteoblast-like MG-63 cells were seeded onto flexible-bottomed plates and subjected to cyclic mechanical stretching (15% elongation, 0.5Hz) for 24 and 48 hours in a Flexercell FX-4000 strain unit. Cellular activities were measured by an assay based on the reduction of the tetrazolium salt, 3-[4,5-dimethyldiazol-2-yl]-2,5-diphenyl tetra-zolium bromide (MTT). The number of viable cells was also determined by the trypan blue dye exclusion technique. Cell cycle progression was checked by flow cytometry. mRNA expressions of apoptosis- and cell cycle-related genes (Bcl2, Bax, cdc2, cdc25C, and cyclin B1) were analyzed using an RT-PCR technique. The number of viable cells significantly decreased in osteoblast-like MG-63 cells subjected to cyclic mechanical stretching for 24 or 48 hours. The MTT activity of stretched cells did not change at 24 hours, whereas a significant decrease was noted at 48 hours in comparison to the unstretched controls. The flow cytometry showed that mechanical stretching induced S-phase cell cycle arrest. Furthermore, exposure to mechanical stretching led to apoptotic cell death, as shown by the increase in the hypodiploid sub-G0/G1 cell population. Furthermore, a decreased cdc25C mRNA level was consistently noted in stretched cells. However, the mRNA expressions of Bcl2, Bax, cdc2, and cyclin B1 genes were not significantly altered compared to the unstretched control cells. High-level mechanical stretching induced S-phase cell cycle arrest and apoptotic cell death in osteoblastic cells. The results suggest that heavy tensional force is a negative regulator of osteoblastic activities and should, therefore, be minimized if bone formation is attempted during orthodontic/orthopedic treatment.

Development of Automatic Stimulating Cell System Using Mechanical Vertical Force

This study developed an automatic monitoring system with a vertical mechanical force to stimulate cancerous cartilage sw1353 cells. The system is controlled by the human-machine interface to stretch cell-cultured substrate continuously which the cells cultured on the PDMS substrate. This system can precisely control the substrate strain, stretch frequency, stretch interval and stopping time when the substrate is stretched. The automatic monitor system can stimulate the cancerous cartilage cells which cultured on the PDMS. In addition, the electrophopresis force was used to measure the cell adhesive ability for the stretched and unstretched cells. The automatic monitoring system with a vertical mechanical force stimulator can stretch substrate which is stretched the strain capacity of substrate ranges from 0.5 to 9%.The stretching frequency ranges from 0.4 to 2.4Hz for this system. The average error of system ranges from 0.033 to 0.034mm after it has conducted 30 consecutive movements. When the cells were conducted 3% substrate strain, 0.8Hz, and 30-minute continuous cycle of tensile strength, the adhesion force of cell ranges from 5.57 to 7.93nN, significantly higher than the unstretched cell which range from 0.92 to 1.25nN.The experimental results showed that the stretching stimulation highly correlates to the cell adhesion.