Dynamic Imaging Reveals Coordinate Effects of Cyclic Stretch and Substrate Stiffness on Endothelial Integrity.
ABSTRACT: We describe an equibiaxial cell stretcher and hybrid, elastic membrane platform designed for dynamic imaging of cells on substrates with physiological stiffness undergoing cyclic stretch. Studies enabled by this device revealed that both substrate stiffness and cyclic stretch coordinately protect pulmonary endothelial monolayers against thrombin-induced disruption. The fluorescence imaging possible with the designed hybrid membranes further revealed similarities and differences in actin and cell dynamics during monolayer recovery. The improved live-cell imaging capabilities of this platform, when used in conjunction with fluorescent probes, will have broad applications for investigations of the impact of biochemical stimuli and mechanotransduction mechanisms on mechanically perturbed tissues.
Project description:Endothelial cell (EC) alignment to directional flow or stretch supports anti-inflammatory functions, but mechanisms controlling polarized structural adaptation in response to physical cues remain unclear. This study aimed to determine whether factors associated with early actin edge ruffling implicated in cell polarization are prerequisite for stress fiber (SF) reorientation in response to cyclic uniaxial stretch. Time-lapse analysis of EGFP-actin in confluent ECs showed that onset of either cyclic uniaxial or equibiaxial stretch caused a non-directional increase in edge ruffling. Edge activity was concentrated in a direction perpendicular to the stretch axis after 60 min, consistent with the direction of SF alignment. Rho-kinase inhibition caused reorientation of both stretch-induced edge ruffling and SF alignment parallel to the stretch axis. Arp2/3 inhibition attenuated stretch-induced cell elongation and disrupted polarized edge dynamics and microtubule organizing center reorientation, but it had no effect on the extent of SF reorientation. Disrupting localization of p21-activated kinase (PAK) did not prevent stretch-induced SF reorientation, suggesting that this Rac effector is not critical in regulating stretch-induced cytoskeletal remodeling. Overall, these results suggest that directional edge ruffling is not a primary mechanism that guides SF reorientation in response to stretch; the two events are coincident but not causal.
Project description:In order to understand the effect of mechanical strain on scleral extracellular matrix remodeling, human scleral fibroblasts were subjected to equibiaxial stretch in vitro and the expression of proteoglycans, metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase-2 (TIMP-2) were evaluated. Isolated human scleral fibroblasts were seeded onto flexible bottom culture plates, and subjected to a cyclic stretch regimen of 15% equibiaxial stretch for 45 s followed by 15s of rest for 6-48 h in the presence of 35SO4. Newly synthesized proteoglycans were measured in the medium by CPC precipitation of radiolabelled glycosaminoglycans. MMP-2 activity and expression levels were measured in the medium by, Western blot, gel zymography and real-time PCR. Steady state levels of TIMP-2 mRNA and membrane-type MMP, MT1-MMP (MMP-14) mRNA were measured in the cell layer using real-time PCR. The predominant gelatinolytic enzyme secreted by scleral fibroblasts was the pro-enzyme form of MMP-2 (ProMMP-2). Mechanical stretch resulted in a significant increase of ProMMP-2 after 12 and 48 h (+76.28%, p<0.05; +19.56%, p<0.01, respectively). Mechanical stretch significantly increased the production of the active form of MMP-2 (ActiveMMP-2) after 48 h (+59.72%, p<0.05) and decreased levels of TIMP-2 mRNA (-22%, p<0.05). The rate of scleral proteoglycan synthesis and the steady state levels of MMP-2 and MMP-14 mRNA were not significantly affected by mechanical stretch. These results suggest that mechanical strain stimulates the activation of MMP-2 by scleral fibroblasts, possibly through increased levels of ProMMP-2 and reduced levels of TIMP-2. Increased levels of ActiveMMP-2 in the sclera would be expected to contribute to scleral extracellular matrix degradation, scleral thinning and possible ocular ectasia.
Project description:Mechanical stimulation is recognized as a potent modulator of cellular behaviors such as proliferation, differentiation, and extracellular matrix assembly. However, the study of how cell-generated traction force changes in response to stretch is generally limited to short-term stimulation. The goal of this work is to determine how cells actively alter their traction force in response to long-term physiological cyclic stretch as a function of cell pre-stress. We have developed, to our knowledge, a novel method to assess traction force after long-term (24 h) uniaxial or biaxial cyclic stretch under conditions of high cell pre-stress with culture on stiff (7.5 kPa) polyacrylamide gels (with or without transforming growth factor ?1 (TGF-?1)) and low pre-stress by treating with blebbistatin or culture on soft gels (0.6 kPa). In response to equibiaxial stretch, valvular interstitial cells on stiff substrates decreased their traction force (from 300 nN to 100 nN) and spread area (from 3000 to 2100 ?m(2)). With uniaxial stretch, the cells had similar decreases in traction force and area and reoriented perpendicular to the stretch. TGF-?1-treated valvular interstitial cells had higher pre-stress (1100 nN) and exhibited a larger drop in traction force with uniaxial stretch, but the percentage changes in force and area with stretch were similar to the non-TGF-?1-treated group. Cells with inhibited myosin II motors increased traction force (from 41 nN to 63 nN) and slightly reoriented toward the stretch direction. In contrast, cells cultured on soft gels increased their traction force significantly, from 15 nN to 45 nN, doubled their spread area, elongated from an initially rounded morphology, and reoriented perpendicular to the uniaxial stretch. Contractile-moment measurements provided results consistent with total traction force measurements. The combined results indicate that the change in traction force in response to external cyclic stretch is dependent upon the initial cell pre-stress. This finding is consistent with depolymerization of initially high-tension actin stress fibers, and reinforcement of an initially low-tension actin cytoskeleton.
Project description:The ability of cells to orient in response to mechanical stimuli is essential to embryonic development, cell migration, mechanotransduction, and other critical physiologic functions in a range of organs. Endothelial cells, fibroblasts, mesenchymal stem cells, and osteoblasts all orient perpendicular to an applied cyclic stretch when plated on stretchable elastic substrates, suggesting a common underlying mechanism. However, many of these same cells orient parallel to stretch in vivo and in 3D culture, and a compelling explanation for the different orientation responses in 2D and 3D has remained elusive. Here, we conducted a series of experiments designed specifically to test the hypothesis that differences in strains transverse to the primary loading direction give rise to the different alignment patterns observed in 2D and 3D cyclic stretch experiments ("strain avoidance"). We found that, in static or low-frequency stretch conditions, cell alignment in fibroblast-populated collagen gels correlated with the presence or absence of a restraining boundary condition rather than with compaction strains. Cyclic stretch could induce perpendicular alignment in 3D culture but only at frequencies an order of magnitude greater than reported to induce perpendicular alignment in 2D. We modified a published model of stress fiber dynamics and were able to reproduce our experimental findings across all conditions tested as well as published data from 2D cyclic stretch experiments. These experimental and model results suggest an explanation for the apparently contradictory alignment responses of cells subjected to cyclic stretch on 2D membranes and in 3D gels.
Project description:Mechanical cues in the cellular environment play important roles in guiding various cell behaviors, such as cell alignment, migration, and differentiation. Previous studies investigated mechanical stretch guided cell alignment pre-dominantly with cyclic stretching whereby an external force is applied to stretch the substrate dynamically (i.e., cyclically) while the cells are attached onto the substrate. In contrast, we created a static pre-stretched anisotropic surface in which the cells were seeded subsequent to stretching the substrate. We hypothesized that the cell senses the physical environment through a more active mechanism, namely, even without external forces the cell can actively apply traction and sense an increased stiffness in the stretched direction and align in that direction. To test our hypothesis, we quantified the extent of pre-stretch induced anisotropy by employing the theory of small deformation superimposed on large and predicted the effective stiffness in the stretch direction as well as its perpendicular direction. We showed mesenchymal stem cells (MSC) aligned in the pre-stretched direction, and the cell alignment and morphology were dependent on the pre-stretch magnitude. In addition, the pre-stretched surface demonstrated an ability to promote early myoblast differentiation of the MSC. This study is the first report on MSC alignment on a statically pre-stretched surface. The cell orientation induced by the pre-stretch induced anisotropy could provide insight into tissue engineering applications involving cells that aligned in vivo in the absence of dynamic mechanical stimuli.
Project description:The goal of this study was to identify microRNAs that are modulated by cyclic stretch. miRNA-Seq was performed comparing samples from cultured E16.5 mouse cardiomyocytes exposed to either cyclic stretch of 16% at 1Hz for 24h or static conditions. Overall design: 3 samples from cardiomyocytes cultured under static conditions were used as the controls. The 3 experimental samples were from cardiomyocytes exposed to cyclic stretch of 16% at 1Hz for 24h.
Project description:The aim of the experiment was to determine the effect of cyclic stretch-relaxation ("stretch") on gene expression patterns in normal diploid human bladder smooth muscle cells. Cells plated on silicone elastomer bottomed 6-well culture dishes were grown to ~80% confluence, serum-depleted for 48h and subjected to cyclic stretch-relaxation at 20% elongation for 4h. Cells seeded in stretch plates but not subjected to stretch served as controls. Total RNA was extracted from both groups of cells, reverse-transcribed, biotin-labeled, fragmented and hybridized to HG-U133A. Four biological replicates were generated for each treatment group (non-stretched or stretched). Keywords = bladder Keywords = smooth muscle cells Keywords = cyclic stretch-relaxation Keywords: other
Project description:The effect of cyclic mecanical stretch on cardiac gene expression was studied in neonatal rat ventricular myocytes (NRVMs). Overall design: The effect of cyclic mecanical stretch on cardiac gene expression was studied in neonatal rat ventricular myocytes (NRVMs) 1, 4, and 12 hours after start of the stretching. There are 5 biological replicates at each timepoint.
Project description:An increase in mechanical load in the heart causes cardiac hypertrophy, either physiologically (heart development, exercise and pregnancy) or pathologically (high blood pressure and heart-valve regurgitation). Understanding cardiac hypertrophy is critical to comprehending the mechanisms of heart development and treatment of heart disease. However, the major molecular event that occurs during physiological or pathological hypertrophy is the dynamic process of sarcomeric addition, and it has not been observed. In this study, a custom-built second harmonic generation (SHG) confocal microscope was used to study dynamic sarcomeric addition in single neonatal CMs in a 3D culture system under acute, uniaxial, static, sustained stretch. Here we report, for the first time, live-cell observations of various modes of dynamic sarcomeric addition (and how these real-time images compare to static images from hypertrophic hearts reported in the literature): 1) Insertion in the mid-region or addition at the end of a myofibril; 2) Sequential addition with an existing myofibril as a template; and 3) Longitudinal splitting of an existing myofibril. The 3D cell culture system developed on a deformable substrate affixed to a stretcher and the SHG live-cell imaging technique are unique tools for real-time analysis of cultured models of hypertrophy.
Project description:Large, elastic arteries buffer the pressure wave originating in the left ventricle and are constantly exposed to higher amplitudes of cyclic stretch (10%) than muscular arteries (2%). As a crucial factor for endothelial and smooth muscle cell function, cyclic stretch has, however, never been studied in ex vivo aortic segments of mice. To investigate the effects of cyclic stretch on vaso-reactivity of mouse aortic segments, we used the Rodent Oscillatory Tension Set-up to study Arterial Compliance (ROTSAC). The aortic segments were clamped at frequencies of 6-600 bpm between two variable preloads, thereby mimicking dilation as upon left ventricular systole and recoiling as during diastole. The preloads corresponding to different transmural pressures were chosen to correspond to a low, normal or high amplitude of cyclic stretch. At different time intervals, cyclic stretch was interrupted, the segments were afterloaded and isometric contractions by ?1-adrenergic stimulation with 2 ?M phenylephrine in the absence and presence of 300 ?M L-NAME (eNOS inhibitor) and/or 35 ?M diltiazem (blocker of voltage-gated Ca2+ channels) were measured. As compared with static or cyclic stretch at low amplitude (<10 mN) or low frequency (0.1 Hz), cyclic stretch at physiological amplitude (>10 mN) and frequency (1-10 Hz) caused better ex vivo conservation of basal NO release with time after mounting. The relaxation of PE-precontracted segments by addition of ACh to stimulate NO release was unaffected by cyclic stretch. In the absence of basal NO release (hence, presence of L-NAME), physiological in comparison with aberrant cyclic stretch decreased the baseline tension, attenuated the phasic contraction by phenylephrine in the absence of extracellular Ca2+ and shifted the smaller tonic contraction more from a voltage-gated Ca2+ channel-mediated to a non-selective cation channel-mediated. Data highlight the need of sufficient mechanical activation of endothelial and vascular smooth muscle cells to maintain basal NO release and low intracellular Ca2+ in the smooth muscle cells in large arteries. Both phenomena may play a vital role in maintaining the high compliance of large arteries.