TY - JOUR
AU1 - Walker,, Matthew
AU2 - Godin,, Michel
AU3 - Pelling, Andrew, E
AB - Abstract Developing methods to study tissue mechanics and myofibroblast activation may lead to new targets for therapeutic treatments that are urgently needed for fibrotic disease. Microtissue arrays are a promising approach to conduct relatively high-throughput research into fibrosis as they recapitulate key biomechanical aspects of the disease through a relevant 3D extracellular environment. In early work, our group developed a device called the MVAS-force to stretch microtissues while enabling simultaneous assessment of their dynamic mechanical behavior. Here, we investigated TGF-β1-induced fibroblast to myofibroblast differentiation in microtissue cultures using our MVAS-force device through assessing α-SMA expression, contractility and stiffness. In doing so, we linked cell-level phenotypic changes to functional changes that characterize the clinical manifestation of fibrotic disease. As expected, TGF-β1 treatment promoted a myofibroblastic phenotype and microtissues became stiffer and possessed increased contractility. These changes were partially reversible upon TGF-β1 withdrawal under a static condition, while, in contrast, long-term cyclic stretching maintained myofibroblast activation. This pro-fibrotic effect of mechanical stretching was absent when TGF-β1 receptors were inhibited. Furthermore, stretching promoted myofibroblast differentiation when microtissues were given latent TGF-β1. Altogether, these results suggest that external mechanical stretch may activate latent TGF-β1 and, accordingly, might be a powerful stimulus for continued myofibroblast activation to progress fibrosis. Further exploration of this pathway with our approach may yield new insights into myofibroblast activation and more effective therapeutic treatments for fibrosis. myofibroblasts, fibrosis, microtissue, TGF-β1, stretch, microfabrication INSIGHT BOX Using a novel high-throughput approach, we quantified the effects of dynamic mechanical stretching on the phenotype and function of cells in 3D microtissue cultures during myofibroblast activation with TGF-β1 treatment and subsequent withdrawal. Our findings demonstrate that mechanical stretch may activate endogenously produced latent TGF-β1 to maintain the presence and activity of myofibroblasts in 3D cultures. Importantly, through this feed forward mechanism, mechanical stretch might be a powerful stimulus that prevents tissues from recovering and promotes the development of fibrosis. However, in contrast to previous reports that investigated the mechanical activation of TGF-β1 in conditions that allow for large cellular perturbations of the extracellular matrix, we found that passive remodeling forces may not be a sufficient stimulus in well-established tissues. INTRODUCTION Myofibroblast activation is a normal healing response following tissue injury found throughout the body, and is essential for rapid wound contraction and de novo matrix deposition [1–5]. However, when unchecked, continued myofibroblast activation may lead to chronic fibrosis and, potentially, a life-threatening loss of tissue functionality [1, 6, 7]. As one of the leading causes of death in developed countries, fibrosis has become an area of high interest for lung, heart, vasculature, liver, renal and eye research [4, 8–11]. Yet the contributing factors that determine whether myofibroblast activation persists and fibrosis progresses into a chronic illness, or whether restoration occurs and tissues regain their initial functionality, remain unclear [4, 11]. Identifying these factors is not only necessary to understand how the activation of myofibroblasts is controlled but, importantly, may also provide valuable insights for future therapeutic approaches to prevent the development and arrest the progression of fibrosis. With that said, transforming growth factor (TGF)-β1, a biochemical inflammatory mediator, is well-accepted to be a key determinant in the development and progression of tissue fibrosis and, as such, has become a central focus of research [12, 13]. In that regard, TGF-β1 treatment is routinely used to induce fibroblast to myofibroblast differentiation in vitro in lieu of any other biochemical stimuli [14, 15], and overexpression of TGF-β1 in animal models consistently exhibit marked fibrotic changes [16, 17]. It is further well recognized that TGF-β1 is a potent agonist of the SMAD2/3 pathway [14], which in fibroblasts leads to α-smooth muscle actin (α-SMA) expression, a commonly used biomarker of myofibroblast differentiation [2, 4, 5]. In addition to canonical SMAD2/3 signaling, TGF-β1 may also activate mitogen-activated protein kinase pathways to further regulate differentiation, proliferation, cell survival and apoptosis [18]. In an autocrine feedforward loop to drive further myofibroblast activation, inactivated TGF-β1 is secreted by myofibroblasts as a large latent complex consisting of the latent TGF-β1 binding protein (LTBP), which associates with the extracellular matrix (ECM), and the latency-associated peptide (LAP), which non-covalently sequesters a TGF-β1 polypeptide and binds to integrins on the cell’s membrane [19–21]. Mechanical stretch, such as encountered in lung tissue from breathing or internally produced by myosin contraction, has been previously proposed as a mechanism to activate this latent source by directly inducing a conformational change in the LAP to release active TGF-β1 into the ECM [19, 22–24]. In doing so, this mechanosensitive pathway may maintain myofibroblast activation to advance the tissue toward of chronic fibrosis. Although mechanical TGF-β1 activation has been extensively studied at the tissue [22], cell [19] and molecular [23, 24] levels, the influence of mechanical stretch on subsequent phenotypic and functional regulation of myofibroblasts has not been clearly demonstrated in vitro with a cellular environment that recapitulates physiological conditions in well-established tissue. Furthermore, while it has been shown that myofibroblast traction forces can liberate active TGF-β1 under conditions of large ECM perturbations, such as thrombin-induced cell contraction [19], it is unclear whether traction forces from passive ECM remodeling in well-established tissues are of a sufficient amplitude or occurrence to influence the regulation of myofibroblast differentiation. Fibrosis and therapeutic treatments have often been investigated in vitro to reduce complexity and increased control; however, the effectiveness of this approach is often hindered by the lack of a biochemically and mechanically relevant environment when using standard 2D cell culture techniques [25, 26]. Instead microtissue arrays, in which cells self-assemble within a collagen ECM around vertical cantilevers into sub-millimeter 3D organized structures, offer an appealing high-throughput alternative [27, 28]. In addition to a relevant ECM, the microtissue method also enables a direct assessment of tissue-level functional changes through tracking the visible deflection of the cantilevers for contractility measurements. Because these abilities, this method has been shown to be especially appropriate for testing the efficacy of fibrosis treatments, as the clinical manifestation of fibrosis is stiff, chronically contracted tissues with a remodeled ECM [28]. In previous work, we developed a microtissue vacuum actuated stretcher (MVAS-force) that was capable of stretching an array of microtissues and simultaneous measurements of dynamic stiffness and contractility [29]. Here, we used our MVAS-force device to link changes in α-SMA expression during TGF-β1 treatment to mechanical changes that are characteristic of tissue fibrosis. We then assessed whether microtissue mechanics and the quiescent fibroblastic phenotype are restored following TGF-β1 withdrawal. Finally, we tested the hypothesis that stretch and passive traction forces may maintain myofibroblast differentiation during TGF-β1 withdrawal through endogenously produced latent TGF-β1 activation. These findings contribute to the field’s understanding of the progression of fibrosis through TGF-β1-induced fibroblast to myofibroblast differentiation. We also established that the MVAS-force device is capable of investigating fibrosis development and, as such, may be a useful tool for pharmacological discovery. Lastly, our findings further implicate that mechanical forces are stimuli that direct tissues away from normal healing pathways and toward fibrotic development, which may have important implications in therapeutic treatment development. METHODS Cell culture Prior to microtissue fabrication, NIH3T3 (ATCC) fibroblast cells were maintained on 100-mm tissue culture dishes (Fisher) at 37|${}^{\circ}\mathrm{C}$| with 5% CO2 until 80–90% confluent in feeder media composed of Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 mg/ml streptomycin and 100 U/ml penicillin antibiotics (all from Hyclone Laboratories Inc.). Microtissue fabrication MVAS-force devices consist of 6 rows of 10 wells, each containing a microtissue formed around a pair of vertical cantilevers. To stretch the microtissues, vacuum chambers border each row and are connected to independent external regulators (SMC ITV0010) controlled through Labview. When a vacuum is applied, the cantilevers closest to the vacuum chamber are actuated, while tissue tension can be measured through the visible deflection in the opposing ‘force-sensing’ cantilevers. MVAS-force devices were fabricated from mold replication steps described previously [29, 30]. Briefly, SU-8 masters for the three layers of the device were created with standard photolithographic techniques. The top layer contains the cell culture wells and vacuum chambers, the middle layer is a thin membrane with the cantilevers, and the bottom layer includes vacuum and empty bottom chambers. To fabricate devices, masters were cast with polydimethylsiloxane (PDMS) containing a 1:10 cross linker ratio and plasma bonded together. Microtissues were cultured in MVAS-force devices as described previously [27, 29, 30]. Prior to seeding, devices were sterilized with 70% ethanol and treated with 0.2% Pluronic F-127 (P6866, Invitrogen) for 2 minutes to reduce cell adhesion; ~650 cells were centrifuged into each well in a solution containing 1.5 mg/ml rat tail collagen type I (354249, Corning), 1× DMEM (SH30003.02, Hyclone), 44 mM NaHCO3, 15 mM d-ribose (R9629, Sigma Aldrich), 1% FBS and 1 M NaOH to achieve a final pH of 7.0–7.4. Excess collagen was removed and devices were incubated at 37°C for 15 min to initiate collagen polymerization. Afterwards, an additional ~130 cells were then centrifuged on top of each tissue. Feeder media was added and changed every 24 hours. Microtissues were allowed to compact and secure themselves to the cantilevers under static conditions with feeder media for 2 days prior to experimentation. Myofibroblast differentiation To assess myofibroblast differentiation, the cell culture media was switched to a differentiation formulation containing DMEM, 5% FBS, 100 mg/ml streptomycin, 100 U/ml penicillin antibiotics and 5 ng/ml TGF-β1 (Peprotech, 100–21). Functional assessment of differentiation was assessed every 24 hours over a 3-day period. Because there was no change to microtissues beyond day 2, differentiation for subsequent experiments was kept to 2 days. To block the differentiation and to assess the contribution of Rho-signaling, the media was supplemented with 1 μM Y27632 (Y27) (Y0503, Sigma Aldrich), a rock inhibitor. We next investigated whether myofibroblast differentiation was reversible, by switching microtissues back to feeder media for 2 days following 2 days of differentiation. Once it was demonstrated that microtissue differentiation was in part reversible, we tested the hypothesis that long-term oscillatory mechanical stretch may maintain myofibroblast differentiation by cyclically loading microtissues at 0.5 Hz 5% strain during dedifferentiation. Further in regards to this hypothesis, to assess whether stretch and passive traction forces activate endogenously and produced TGF-β1, TGF-β1 receptors were blocked with 10 μM GW788388 (GW) (16255–1, Cayman Chemicals) during dedifferentiation. Lastly, differentiation was assessed with a latent source of 5 ng/ml TGF-β1 (299-LT-005, R&D Systems) again under both static and stretching conditions. Although this latent source is not bound to LTBP, this treatment permitted us to report on the mechanical regulation of the small latent complex containing TGF-β1 and LAP. Functional assessment of differentiation To assess how microtissues are functionally altered during myofibroblast differentiation, microtissue mechanics were assessed in situ using the MVAS-force at 37°C and under 5% CO2 [29]. Briefly, forces were measured through the visible deflection of the force-sensing cantilever and its known spring constant. To calculate the deflection, images were captured at focal planes containing both the top and bottom of the cantilevers. The bottom positions of the cantilevers were measured using a centroid algorithm, while the tops were tracked using pattern matching with adaptive template learning in Labview. Static deflections were calculated by subtracting the top and bottom positions and gave an assessment of the resting contractility. To assess stiffness changes, the dynamic mechanical behaviors were measured at 0.5 Hz with 3% strain. Images at both the tops and bottoms of the cantilevers were captured at 15fps for a minute. Tissue tension was calculated from the difference in the positions of the force-sensing cantilever in those images after accounting for the phase shift caused by the camera delay between capturing the two focal planes. Microtissue strain (ε) was defined as the percent change in length measured from the innermost edges of the tops of the cantilevers (equation 1). The storage stiffness (k’), a measurement of elasticity, was then calculated as the ratio of the magnitudes of the Fourier transforms of tension and strain multiplied by the cosine of the phase lag between force and strain (equation 2). $$\begin{equation} \varepsilon (t)=\frac{\mathrm{length}(t)-{\mathrm{length}}_o}{{\mathrm{length}}_o}\times 100 \end{equation}$$(1) $$\begin{equation} {k}^{\prime }={\left\{\frac{\left|\mathrm{FFT}\left[\mathrm{tension}(t)\right]\right|}{\left|\mathrm{FFT}\left[\varepsilon (t)\right]\right|}\right\}}_{f_o}\mathit{\cos}\ \delta . \end{equation}$$(2) Phenotypic assessment of differentiation As in previous work [2, 4, 5], α-SMA was used as biomarker to assess myofibroblast differentiation and imaged with standard immunofluorescence techniques. Briefly, microtissues were fixed and permeabilized in situ with ice-cold methanol for 10 min. To prevent non-specific binding, microtissues were blocked for 30 min with 5% FBS. α-SMA was labeled with 1:100 primary antibody produced in rabbit (Abcam, ab5694) at room temperature for 2 hours and then 1:200 Goat anti-rabbit IgG secondary antibody conjugated to Alexa Fluor 488 (Invitrogen, A11034) at room temperature for an addition 2 hours. Cell nuclei were stained with DAPI (Fisher, D1306). Image stacks of microtissues were acquired on a TiE A1-R laser scanning confocal microscope (Nikon) with appropriate laser lines and filter blocks. To produce heat maps, images were flattened by integration, spatially aligned and averaged together. α-SMA expression was normalized to the nuclei fluorescence to give a per cell measurement and to account for differences in proliferation. Data analysis and statistics All numerical data are presented as mean ± standard error. Statistical tests, as described in the results, were performed using Originlab 8.5 (Northampton, MA) with P < 0.05 considered statistically significant. RESULTS Myofibroblast differentiation in microtissue cultures Myofibroblast differentiation has historically been characterized through biochemical assays of biomarker expression (i.e. α-SMA) [2, 4, 5] in cells grown in 2D culture. Comparably, there have been far fewer in vitro investigations that have linked the appearance of a myofibroblastic phenotype to functional changes in mechanical behavior (i.e. increased stiffness and contractility) at the tissue-level, which are characteristic of fibrosis [28, 31]. For that reason, we assessed both changes to static contractility and dynamic stiffness of microtissues during myofibroblast differentiation using our MVAS-force device (Fig. 1). Furthermore, while other work has evaluated TGF-β treatment in microtissues immediately following cell seeding [28, 32], our goal here was to investigate myofibroblast differentiation in well-established tissue. As such, we allowed microtissues to compact under static conditions for 2 days prior to any experimentation. In close agreement with previous work [27, 33], microtissues generated an average (N = 23) resting contractility of 5.2 ± 0.5 μN and a storage stiffness of 1.06 ± 0.05 μN/%, and at this time (Experimental Day 0), there were no differences between treatment and control groups (t-test P > 0.05). To account for variation between samples, all subsequent measurements are reported as a delta from their respective baseline value measured at Experimental Day 0. Figure 1 Open in new tabDownload slide TGF-β1 treatment contracts and stiffens microtissue cultures. At Experimental Day 0, once microtissues had compacted and were securely anchored to the cantilevers (2 days after seeding), they were either switched to a media containing TGF-β1 to promote myofibroblast differentiation or kept in normal feeder for an additional 3 days (a). TGF-β1 treatment increased the resting contractility compared with feeder media indicated by a greater deflection in the force-sensing cantilever (b). Additionally, the dynamic stiffness of microtissues was measured in situ with the MVAS-force at 0.5 Hz. Representative clockwise-oriented tension-strain loops with normal feeder and differentiation media are in (c) and (d), respectively. While there was no difference in stiffness between Experimental Days 0 and 3 with feeder media, the stiffness markedly increased with TGF-β1 treatment. The average changes to the resting tension and stiffness over the 3-day period are in (e) and (f), respectively. The TGF-β1 induced increased tension and stiffness largely occurred over the first 2 days, leveling off by Day 3. The effects of TGF-β1 treatment were completely blocked with concurrent treatment with y27, a ROCK inhibitor, indicating the role of Rho-induced stress fiber formation and myosin contraction during myofibroblast differentiation (g and h). The scale bars in (a) and (b) represent 100 and 50 μm, respectively. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 1 Open in new tabDownload slide TGF-β1 treatment contracts and stiffens microtissue cultures. At Experimental Day 0, once microtissues had compacted and were securely anchored to the cantilevers (2 days after seeding), they were either switched to a media containing TGF-β1 to promote myofibroblast differentiation or kept in normal feeder for an additional 3 days (a). TGF-β1 treatment increased the resting contractility compared with feeder media indicated by a greater deflection in the force-sensing cantilever (b). Additionally, the dynamic stiffness of microtissues was measured in situ with the MVAS-force at 0.5 Hz. Representative clockwise-oriented tension-strain loops with normal feeder and differentiation media are in (c) and (d), respectively. While there was no difference in stiffness between Experimental Days 0 and 3 with feeder media, the stiffness markedly increased with TGF-β1 treatment. The average changes to the resting tension and stiffness over the 3-day period are in (e) and (f), respectively. The TGF-β1 induced increased tension and stiffness largely occurred over the first 2 days, leveling off by Day 3. The effects of TGF-β1 treatment were completely blocked with concurrent treatment with y27, a ROCK inhibitor, indicating the role of Rho-induced stress fiber formation and myosin contraction during myofibroblast differentiation (g and h). The scale bars in (a) and (b) represent 100 and 50 μm, respectively. *P < 0.05, **P < 0.01, ***P < 0.001. As expected, TGF-β1 treatment increased the resting tension and stiffness over a 3-day period compared with the control group (Fig. 1e and f, respectively) (t-tests). Functional changes largely occurred over the first 2 days of treatment, with no significant difference to either contractility or stiffness between Days 2 and 3 (repeated measures t-tests P > 0.05). For this reason, 2 days was used as the time point for all subsequent experiments. After 2 days of TGF-β1 treatment, there was a 4.4 ± 0.4μN increase in contractility and a 93 ± 10% increase in stiffness. Inhibiting Rho-signaling with the ROCK inhibitor y27 blocked TGF-β1-induced functional changes (Fig. 1g and h). In fact, concurrent treatment of TGF-β1 with y27 reduced microtissue contractility and stiffness below control microtissues maintained in feeder media (P < 0.05 and P < 0.01, one-way ANOVA). These results are not overly surprising as Rho is known to direct the assembly and stabilization of the actin cytoskeleton [34] and control contractility through the deactivation of myosin phosphatase and phosphorylation of myosin light chain [35]. In addition, several studies have shown the importance of Rho/ROCK signaling in controlling collagen synthesis [36–38], which could also have contributed to the decreased microtissue stiffness with y27. To link functional changes in microtissues to a myofibroblast phenotype change, α-SMA was immunofluorescently imaged. Average heat maps of the distribution of cells and α-SMA expression indicated that TGF-β1 treatment lead to myofibroblast differentiation predominately in cells directly associating with the relatively stiff PDMS cantilevers and around the perimeter of the tissue (Fig. 2a). Moreover, the distribution of cells in the tissues became less uniform as TGF-β1 treatment increased the concentration of cells toward the center of the tissue. In contrast, however, there was no change to the total microtissue width on bright field images (data not shown, P > 0.05, t-test). In comparison to TGF-β1 alone, the spatial distribution of cells was not as affected with the concurrent treatment of TGF-β1 and y27. Figure 2 Open in new tabDownload slide TGF-β1 induces myofibroblast differentiation of microtissues. To quantify TGF-β1-induced myofibroblast differentiation, microtissues were stained for α-SMA. Average heat maps of cell nuclei, α-SMA expression and normalized expression are in (a). Total α-SMA expression normalized to the number of cells is in (b). TGF-β1 treatment significantly increased α-SMA expression compared with the control. Concurrent treatment with y27 still had greater α-SMA expression than control but slightly, albeit not significantly, less than TGF-β1 alone. Images of representative centrally located magnified regions are in (c). TGF-β1 treatment promoted the expression and polymerization of dense α-SMA fibers. In microtissues that concomitantly received y27, α-SMA was not as densely polymerized into organized fibers, indicating the role of Rho in the recruitment of α-SMA into stress fibers. The scale bars in (a) and (c) represent 100 and 50 μm, respectively. **P < 0.01, ***P < 0.001. Figure 2 Open in new tabDownload slide TGF-β1 induces myofibroblast differentiation of microtissues. To quantify TGF-β1-induced myofibroblast differentiation, microtissues were stained for α-SMA. Average heat maps of cell nuclei, α-SMA expression and normalized expression are in (a). Total α-SMA expression normalized to the number of cells is in (b). TGF-β1 treatment significantly increased α-SMA expression compared with the control. Concurrent treatment with y27 still had greater α-SMA expression than control but slightly, albeit not significantly, less than TGF-β1 alone. Images of representative centrally located magnified regions are in (c). TGF-β1 treatment promoted the expression and polymerization of dense α-SMA fibers. In microtissues that concomitantly received y27, α-SMA was not as densely polymerized into organized fibers, indicating the role of Rho in the recruitment of α-SMA into stress fibers. The scale bars in (a) and (c) represent 100 and 50 μm, respectively. **P < 0.01, ***P < 0.001. Overall, the total normalized α-SMA expression increased by 120% with TGF-β1 treatment compared with the control (Fig. 2b) (one-way ANOVA, P < 0.001). Furthermore, representative images of centrally located regions show that TGF-β1 treatment caused α-SMA to be expressed in polymerized fibers (Fig. 2c). α-SMA expression was also increased with the concurrent treatment of TGF-β1 and y27 compared with the control (P < 0.01). However, compared with TGF-β1 alone, y27 treatment seemed to have decreased α-SMA expression, albeit the difference was not statistically significant (P = 0.08). Also in microtissues that concomitantly received y27, α-SMA was less densely polymerized into organized fibers than microtissues treated with TGF-β1 only. These findings demonstrate the involvement of Rho/ROCK pathways in the recruitment of α-SMA into stress fibers. Mechanical stretch sustains myofibroblast differentiation The initial appearance of myofibroblasts is a normal physiological response following injury, contributing to wound contraction and collagen matrix deposition [1–5]. In tissues that are fully repaired, the myofibroblast phenotype disappears from the wound space and tissues regain their initial functionality. For reasons unknown, however, the presence of myofibroblasts may linger beyond normal wound healing, leading to a loss of tissue functionality and chronic fibrosis [1, 6, 7]. Accordingly, we assessed whether microtissues may recover their initial fibroblastic phenotype and functionality if the TGF-β1 treatment was withdrawn following 2 days of differentiation. Switching microtissues back to feeder media for 2 days decreased contractility by −2.1 ± 0.3 μN and stiffness by −18 ± 3% (one-way ANOVA, P < 0.01 and P < 0.001) (Fig. 3a and b). Removing TGF-β1 also reduced α-SMA expression by 40 ± 4% (one-way ANOVA, P < 0.001) (Fig. 3c–e). Importantly, these findings show that the presence and functionality of myofibroblasts can in part be reversed in microtissues that were differentiated as in a normal wound healing process. Figure 3 Open in new tabDownload slide Myofibroblast differentiation is, in part, reversible but sustained through mechanical stretch. To assess whether the activation of the myofibroblast phenotype could be reversed, microtissues were allowed to compact for 2 days in feeder media, then differentiated with TGF-β1 media for 2 days and finally switched back to feeder media. Compared with microtissues that were sustained in differentiation media, TGF-β1 withdrawal decreased resting tension and stiffness (a and b, respectively). In contrast, however, the tension and stiffness did not decrease in microtissues that were dynamically stretched at 5% 0.5 Hz in addition to TGF-β1 withdrawal. These changes were mirrored in heat maps, normalized total, and representative images of α-SMA expression (c–e, respectively). TGF-β1 withdrawal decreased α-SMA expression and fiber formation. In contrast, dynamic stretching, in part, prevented the loss of myofibroblast phenotype but was still significantly reduced compared with continued TGF-β1-treated microtissues. These findings indicate that mechanical stretch may sustain myofibroblast differentiation. The scale bars in (c) and (e) represent 100 and 50 μm, respectively. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 3 Open in new tabDownload slide Myofibroblast differentiation is, in part, reversible but sustained through mechanical stretch. To assess whether the activation of the myofibroblast phenotype could be reversed, microtissues were allowed to compact for 2 days in feeder media, then differentiated with TGF-β1 media for 2 days and finally switched back to feeder media. Compared with microtissues that were sustained in differentiation media, TGF-β1 withdrawal decreased resting tension and stiffness (a and b, respectively). In contrast, however, the tension and stiffness did not decrease in microtissues that were dynamically stretched at 5% 0.5 Hz in addition to TGF-β1 withdrawal. These changes were mirrored in heat maps, normalized total, and representative images of α-SMA expression (c–e, respectively). TGF-β1 withdrawal decreased α-SMA expression and fiber formation. In contrast, dynamic stretching, in part, prevented the loss of myofibroblast phenotype but was still significantly reduced compared with continued TGF-β1-treated microtissues. These findings indicate that mechanical stretch may sustain myofibroblast differentiation. The scale bars in (c) and (e) represent 100 and 50 μm, respectively. *P < 0.05, **P < 0.01, ***P < 0.001. Mechanosensing pathways have been previously identified as key stimuli in myofibroblast differentiation [3, 5, 39, 40]; however, the role of dynamic stretch remains unclear. In that regard, stretching microtissues during TGF-β1 treatment produced no functional or phenotypic differences (Supplementary Information 1). In contrast, however, stretching microtissues upon TGF-β1 withdrawal prevented the decrease in contractility and stiffness that had been observed in static cultures that were also switched back to feeder media. In fact, stretching with TGF-β1 withdrawal sustained microtissue mechanics to the same extent as continued TGF-β1 treatment (P > 0.05). This maintenance of microtissue mechanics was also mirrored by an increased α-SMA expression compared with static conditions (one-way ANOVA P < 0.05). Admittedly, however, the expression was still lower than in microtissues kept in TGF-β1 (P < 0.05). Nevertheless, these results suggest that cyclic stretching may in part be responsible for the maintenance of myofibroblastic phenotype, leading to chronic fibrosis. Mechanical stretch activates latent TGF-β1 in microtissues It has been previously hypothesized that dynamic stretching may contribute to myofibroblast differentiation through activating latent TGF-β1 sources sequestered by matrix proteins. This latent form can be produced and secreted into the extracellular environment by myofibroblasts in a feedforward loop, to drive further differentiation [19–21]. Therefore, to assess whether mechanical stretching sustained myofibroblast differentiation through activating endogenous TGF-β1, we attempted to dedifferentiate microtissues, while TGF-β1 receptors were blocked with GW to inhibit autocrine signaling. As expected, blocking receptors did not affect the observed decrease to the stiffness or α-SMA expression compared with TGF-β1 withdrawal only (one-way ANOVA, P > 0.05) (Fig. 4a and c). Furthermore, when receptors were blocked, microtissue stiffness and α-SMA expression, distribution and organization were no different under static and stretching conditions (one-way ANOVA, P > 0.05) (Fig. 4 a–d). These results indicate that stretch maintains myofibroblast functionality and phenotype through autocrine TGF-β1 signaling. Figure 4 Open in new tabDownload slide TGF-β1 receptor inhibition prevents sustained myofibroblast differentiation through mechanical stretch. To assess whether dynamic stretching sustained myofibroblast differentiation through activation of endogenous latent TGF-β1 in an autocrine signaling pathway, TGF-β1 receptors were blocked with GW during exogenous TGF-β1 withdrawal. Upon blocking TGF-β1 receptors, dynamic stretching did not affect the reduction of microtissue stiffness (a) or α-SMA expression (b–d) that accompanied exogenous TGF-β1 withdrawal. These findings suggest that the sustained differentiation with mechanical stretching is through endogenous TGF-β1 activation. The scale bars in (b) and (d) represent 100 and 50 μm, respectively. Figure 4 Open in new tabDownload slide TGF-β1 receptor inhibition prevents sustained myofibroblast differentiation through mechanical stretch. To assess whether dynamic stretching sustained myofibroblast differentiation through activation of endogenous latent TGF-β1 in an autocrine signaling pathway, TGF-β1 receptors were blocked with GW during exogenous TGF-β1 withdrawal. Upon blocking TGF-β1 receptors, dynamic stretching did not affect the reduction of microtissue stiffness (a) or α-SMA expression (b–d) that accompanied exogenous TGF-β1 withdrawal. These findings suggest that the sustained differentiation with mechanical stretching is through endogenous TGF-β1 activation. The scale bars in (b) and (d) represent 100 and 50 μm, respectively. In the latent form, TGF-β1 is non-covalently caged by the mechanosensitive LAP, which effectively decreases its bioavailability. It has been hypothesized that stretch may cause conformational changes to the LAP to free TGF-β1 to promote continued myofibroblast differentiation. Therefore, to assess whether dynamic stretch was capable of liberating active TGF-β1 from LAP, microtissues were treated with 5 ng/ml Latent TGF-β1 for 2 days. Latent TGF-β1 alone produced no change in the stiffness (repeated measures t-test, P > 0.05) and α-SMA expression remained low throughout microtissues (Fig. 5). In contrast, dynamic stretch, in addition to latent TGF-β1, increased microtissue stiffness and α-SMA expression (t-tests, P < 0.001). Admittedly, the stiffness and expression levels still remained lower than if the same concentration of activated TGF-β1 was given, likely because stretch did not activate 100% of the latent TGF-β1 given to the microtissues. Nevertheless, these results indicate that stretch can mechanically activate latent TGF-β1. Figure 5 Open in new tabDownload slide Latent TGF-β1 is activated by mechanical stretch to promote myofibroblast differentiation. To demonstrate that a latent source of TGF-β1 can be activated by mechanical stretch, myofibroblast differentiation was assessed under static and stretching conditions with exogenous latent TGF-β1. Dynamic stretch significantly increased microtissue stiffness (a) and α-SMA expression (c–d) compared with static conditions. These findings indicate that mechanical stretching can activate latent TGF-β1, further supporting its role in sustaining myofibroblast differentiation during TGF-β1 withdrawal. The scale bars in (b) and (d) represent 100 and 50 μm, respectively. ***P < 0.001. Figure 5 Open in new tabDownload slide Latent TGF-β1 is activated by mechanical stretch to promote myofibroblast differentiation. To demonstrate that a latent source of TGF-β1 can be activated by mechanical stretch, myofibroblast differentiation was assessed under static and stretching conditions with exogenous latent TGF-β1. Dynamic stretch significantly increased microtissue stiffness (a) and α-SMA expression (c–d) compared with static conditions. These findings indicate that mechanical stretching can activate latent TGF-β1, further supporting its role in sustaining myofibroblast differentiation during TGF-β1 withdrawal. The scale bars in (b) and (d) represent 100 and 50 μm, respectively. ***P < 0.001. DISCUSSION Continued research and development of techniques are urgently needed to further our understanding of myofibroblast activation and to discover new drug targets to treat fibrosis. Not only do microtissues recapitulate key biochemical and mechanical properties of both healthy and fibrotic tissues, but also they allow cell-level phenotypic and tissue-level functional screening in a relatively high-throughput manner. For these reasons, microtissues are a compelling approach to conduct fibrosis research [28]. Here, we aimed to investigate myofibroblast activation and deactivation under cyclic stretching in a microtissue model using our MVAS-force device. Importantly, this approach allowed us to link changes to protein expression to mechanical behaviors that directly reflect the clinical manifestation of fibrotic disease. As expected, TGF-β1 treatment led to fibroblast to myofibroblast differentiation, indicated by α-SMA expression and increased microtissue contractility and stiffness. These changes were consistent with previous work that assessed microtissues populated with human lung fibroblasts [28]. With that said, human lung fibroblast microtissues generated much greater forces than the embryonic fibroblast cell line we used here, and previously reported by Legant et al. (2009). Our findings are further in accordance with multiple studies that have shown that increased tissue stiffness is a characteristic feature of fibrosis, albeit the changes that we observed over a relatively short experimental time were less than those previously reported in fibrotic lungs (increases from ~2 to ~17 kPa) [41–43] and liver (~1 to 3–22 kPa) [44]. In organs with established fibrosis, stiffness changes are thought to arise from extracellular protein deposition, particularly fibrillar collagen, and matrix crosslinking [44, 45]. Increased deposition of collagen fibers upon myofibroblast activation has already been reported in microtissues [28] and thus likely contributed to our results. Altered cell (as opposed to matrix) stiffness may also have made a significant contribution to the change in tissue mechanics. In that regard, it has been previous argued that differences between decellularized matrices cannot fully account for differences in stiffness between normal and fibrotic tissue samples [41]. Although the contribution from altered cell properties to fibrotic tissue stiffness has yet to be established, it may be considerable [7]. A stiffer ECM is well accepted to encourage myofibroblast activation through mechanosensing at focal adhesions and Rho/ROCK signaling [39, 40]. Therefore, it was not surprising that we found that concurrent ROCK inhibition during TGF-β1 treatment prevented fibrotic functional changes and helped to preserve the quiescent fibroblastic phenotype. Accordingly, the Rho/ROCK pathway has become an attractive target for anti-fibrotic pharmaceutical treatments [46–48], and from our demonstration here, the MVAS-force may offer a method for testing and developing such treatments in tissue-like structures prior to clinical trials. In addition to mechanosensing through focal adhesions, α-SMA itself is beginning to be considered mechanosensitive, as it only localizes to stress fibers when cells are subjected to considerable mechanical loads [3]. For example, α-SMA expression in stress fibers requires a substrate stiffness around 20 kPa and reducing cellular tension by inhibiting myosin contraction leads to disassembly of α-SMA stress fibers [49, 50]. This could provide a direct explanation as to the tight coupling between tissue mechanics and α-SMA expression that we generally observed outside of ROCK inhibition. Although it is generally agreed that increased substrate stiffness favors fibroblast differentiation into myofibroblasts [51, 52], the role of cyclic mechanical stretch, as cells would encounter in airways and vasculature, is less clear. There have been conflicting reports as to whether cyclic stretch promotes myofibroblast differentiation [53–55] or serves as a protective role promoting a quiescent fibroblastic phenotype [56, 57]. In contrast to those reports, we did not find that stretch had any effect on myofibroblast activation in microtissues. Besides differences in cell types, the ECM stiffness may partially explain these conflicting reports. In that regard, stretching of lung fibroblasts cultured on stiff (30 kPa) gels has been shown to augment myofibroblastic phenotype, while stretching on soft (2 kPa) gels had no affect [55]. Differences in the strain field may also contribute as enhanced anisotropy when under biaxial loading has been shown to augment myofibroblast activation from valve interstitial cells [54]. Following stimulation with TGF-β1, we found that subsequent withdrawal partially reversed the myofibroblastic expression phenotype and mechanical behavior. Classically in the paradigm for tissue repair, terminally differentiated myofibroblasts are removed from healing tissue through apoptosis [58]. However, opposed to earlier investigations which suggested that TGF-β1 induces a relatively stable alteration in cell phenotype [14], more recent work has found that myofibroblasts have the capacity for de-differentiation back into a quiescent fibroblast phenotype [31, 59–61]. Perhaps of more significance, in contrast to microtissues kept under static conditions, we found that cyclic stretching maintained the myofibroblastic phenotype and tissue mechanics during TGF-β1 withdrawal. This finding can likely be accounted by autocrine signaling involving mechanical activation of latent TGF-β1 since blocking TGF-β1 receptors while stretching led to identical behaviors as in static conditions. We further demonstrated that stretching microtissues could activate latent TGF-β1 to promote differentiation toward a myofibroblast phenotype and to increase tissue stiffness. Of note, the recombinant source of latent TGF-β1 lacked LTBP. While previous work has shown that a mutant form of TGF-β1 unable to bind LTBP can still be activated, it does so ineffectively [62]. As such, we only saw a modest degree of myofibroblast differentiation with this treatment when mechanically conditioned. With that said, the activation mechanism without LTBP is not currently well understood, and in regards to the current paradigm of mechanical activation, LTBP is a necessary tether between the LAP and the ECM [20]. Consequently, there are three possible explanations for our finding. Firstly, other work has shown that cells can secrete free LTBP [21], and therefore, LTBP may have combined with the exogenous latent TGF-β1 outside of the cell. However, we are unaware of any literature in support for this postulation and it is unlikely that the ECM contains the correct enzyme for catalyzing the di-sulphide bond. Secondly, the cells themselves may have simply secreted latent TGF-β1 with LTBP, which then could have been activated upon stretching the cultures. A last, possible explanation is that protease digestion of LAP by matrix metallopeptidase (MMP)-2,-9 may be mechanically regulated by stretch in tissues. In support of this hypothesis, much work has already tied mechanical forces to MMP activity [63]. Future work remains in further investigating these possibilities and determining the mechanisms of activation of TGF-β1 without LTBP. Nevertheless, our finding that stretch maintains myofibroblast differentiation upon TGF-β1 withdrawal adds to an already large precedent that autocrine TGF-β1 signaling is mechanically regulated. For example, tensile loading of fibrotic lung strips have been shown to release active TGF-β1 and subsequently increase Smad2/3 signaling [22]. Furthermore, single molecule force spectroscopy to pull directly on LAP has shown to induce a conformation change that frees active TGF-β1 in a purely mechanical process [23, 24]. In the same manner as external stretch, contractile forces from lung fibroblasts have also been shown to directly activate latent TGF-β1 from the ECM independent of proteolytic activity [19]. In this regard, large ECM perturbations from both a thrombin-induced contraction and as well as during a 24-hour period immediately following cell seeding were shown to liberate TGF-β1. In contrast to this previous report, however, we did not observe any difference in myofibroblast deactivation with and without a TGF-β1 receptor block under static conditions, and exogenous latent TGF-β1 treatment had no effect on static cultures. Altogether, these results indicate that the activation of latent TGF-β1 did not contribute to responses under static culturing. Therefore, although large ECM perturbations produced through contractile forces from myofibroblasts during wound contraction and matrix compaction are thought to be strong enough to open up the LAP to liberate active TGF-β1 [19], passive remodeling forces may not be or at least do not happen at an appreciable occurrence to significantly change myofibroblast activation in well-established microtissues. On the other hand, external stretching that simulates breathing in the lung and pulsatile flow in vasculature is capable of maintaining myofibroblast behavior in microtissues through an autocrine-signaling pathway. CONCLUSIONS In this article, we demonstrated the effectiveness of the MVAS-force device to investigate fibrosis by showing that microtissues are able to recapitulate characteristic mechanical and biological features that occur in fibrotic disease, namely increased contractility, stiffness and α-SMA expression. These changes were partially reversible upon exogenous TGF-β1 withdrawal. In addition to its capability for relatively high-throughput mechanical assessment of tissue-level mechanics, the MVAS-force also permitted long-term conditioning of fibrotic microtissues to mimic the stretch cells experience in lung and vasculature tissue. Importantly, stretch maintained myofibroblast activation following exogenous TGF-β1 withdrawal likely through endogenous latent TGF-β1 activation. Therefore, external mechanical stretch might be a powerful stimulus for continued myofibroblast activation that progresses fibrotic development. However, unlike previous work that investigated large ECM perturbations following a wound, our findings suggest that this mechanosensitive pathway is not sufficiently responsive to cellular traction forces in well-established tissues and thus, in this manner, may not influence myofibroblast differentiation in later stages of fibrosis. To conclude, further research into pathways linking mechanical forces to soluble mediators may have important implications in the development of therapeutic treatments urgently needed to stop the advancement of fibrosis. ACKNOWLEDGEMENTS M.W. is supported by OGS (Ontario Graduate Scholarship). The authors acknowledge support from individual NSERC Discovery Grants (M.G. and A.E.P.). M.G. and A.E.P. acknowledge the Canadian Foundation for Innovation. FUNDING Ontario Graduate Scholarship, NSERC Discovery Grants, Canadian Foundation for Innovation. CONFLICTS OF INTEREST STATEMENT There are no conflicts to declare. DATA AVAILABILITY The data generated during the current study is available from the corresponding author upon reasonable request. REFERENCES 1. Hinz B . The myofibroblast in connective tissue repair and regeneration. In: Regenerative Medicine and Biomaterials for the Repair of Connective Tissues . Amsterdam, Netherlands : Elsevier , 2010 , 39 – 80 . Google Scholar Crossref Search ADS Google Scholar Google Preview WorldCat COPAC 2. Desmoulière A , Chaponnier C, Gabbiani G. Tissue repair, contraction, and the myofibroblast . Wound Repair and Regeneration. 2005 ; 13 : 7 – 12 . Google Scholar Crossref Search ADS WorldCat 3. Hinz B . Formation and function of the myofibroblast during tissue repair . Journal of Investigative Dermatology. 2007 ; 127 : 526 – 37 . Google Scholar Crossref Search ADS WorldCat 4. Hinz B , Phan SH, Thannickal VJ et al. The myofibroblast: One function . multiple origins. Am J Pathol. 2007 ; 170 : 1807 – 16 . Google Scholar Crossref Search ADS WorldCat 5. Tomasek JJ , Gabbiani G, Hinz B et al. Myofibroblasts and mechano: Regulation of connective tissue remodelling . Nature Reviews Molecular Cell Biology. 2002 ; 3 : 349 – 63 . Google Scholar Crossref Search ADS PubMed WorldCat 6. Darby IA , Hewitson TD. Fibroblast differentiation in wound healing and fibrosis . Int Rev Cytol. 2007 ; 257 : 143 – 79 . Google Scholar Crossref Search ADS PubMed WorldCat 7. Wells RG . Tissue mechanics and fibrosis . Biochimica et Biophysica Acta - Molecular Basis of Disease. 2013 ; 1832 : 884 – 90 . Google Scholar Crossref Search ADS WorldCat 8. Gabbiani G . The myofibroblast in wound healing and fibrocontractive diseases . J Pathol. 2003 ; 200 : 500 – 3 . Google Scholar Crossref Search ADS PubMed WorldCat 9. Friedlander M . Fibrosis and diseases of the eye . Journal of Clinical Investigation. 2007 ; 117 : 576 – 86 . Google Scholar Crossref Search ADS WorldCat 10. Travers JG , Kamal FA, Robbins J et al. Cardiac fibrosis: The fibroblast awakens . Circulation Research. 2016 ; 118 : 1021 – 40 . Google Scholar Crossref Search ADS PubMed WorldCat 11. Myofibroblasts HB . Exp Eye Res. 2016 ; 142 : 56 – 70 . Crossref Search ADS PubMed 12. Biernacka A , Dobaczewski M, Frangogiannis NG. TGF-β signaling in fibrosis . Growth Factors. 2011 ; 29 : 196 – 202 . Google Scholar Crossref Search ADS PubMed WorldCat 13. Meng XM , Nikolic-Paterson DJ, Lan HY. TGF-β: The master regulator of fibrosis . Nature Reviews Nephrology. 2016 ; 12 : 325 – 38 . Google Scholar Crossref Search ADS PubMed WorldCat 14. Evans RA , Tian YC, Steadman R et al. TGF-β1-mediated fibroblast-myofibroblast terminal differentiation - the role of Smad proteins . Exp Cell Res. 2003 ; 282 : 90 – 100 . Google Scholar Crossref Search ADS PubMed WorldCat 15. Desmouliere A , Geinoz A, Gabbiani F et al. Transforming growth factor-β1 induces α-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts . J Cell Biol. 1993 ; 122 : 103 – 11 . Google Scholar Crossref Search ADS PubMed WorldCat 16. Sime PJ , Xing Z, Graham FL et al. Adenovector-mediated gene transfer of active transforming growth factor- β1 induces prolonged severe fibrosis in rat lung . J Clin Invest. 1997 ; 100 : 768 – 76 . Google Scholar Crossref Search ADS PubMed WorldCat 17. Zhao J , Sime PJ, Bringas P et al. Spatial-specific TGF-beta1 adenoviral expression determines morphogenetic phenotypes in embryonic mouse lung . Eur J Cell Biol. 1999 ; 78 : 715 – 25 . Google Scholar Crossref Search ADS PubMed WorldCat 18. Zhang YE . Non-Smad pathways in TGF-β signaling . Cell Research. 2009 ; 19 : 128 – 39 . Google Scholar Crossref Search ADS PubMed WorldCat 19. Wipff PJ , Rifkin DB, Meister JJ et al. Myofibroblast contraction activates latent TGF-β1 from the extracellular matrix . J Cell Biol. 2007 ; 179 : 1311 – 23 . Google Scholar Crossref Search ADS PubMed WorldCat 20. Annes JP , Munger JS, Rifkin DB. Making sense of latent TGFβ activation . Journal of Cell Science. 2003 ; 116 : 217 – 24 . Google Scholar Crossref Search ADS PubMed WorldCat 21. Miyazono K , Olofsson A, Colosetti P et al. A role of the latent TGF-beta 1-binding protein in the assembly and secretion of TGF-beta 1 . EMBO J. 1991 ; 10 : 1091 – 101 . Google Scholar Crossref Search ADS PubMed WorldCat 22. Froese AR , Shimbori C, Bellaye P-S et al. Stretch-induced activation of transforming growth factor-β 1 in pulmonary fibrosis . Am J Respir Crit Care Med. 2016 ; 194 : 84 – 96 . Google Scholar Crossref Search ADS PubMed WorldCat 23. Buscemi L , Ramonet D, Klingberg F et al. The single-molecule mechanics of the latent TGF-β1 complex . Curr Biol. 2011 ; 21 : 2046 – 54 . Google Scholar Crossref Search ADS PubMed WorldCat 24. Shi M , Zhu J, Wang R et al. Latent TGF-β structure and activation . Nature. 2011 ; 474 : 343 – 51 . Google Scholar Crossref Search ADS PubMed WorldCat 25. Edmondson R , Broglie JJ, Adcock AF et al. Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors . Assay Drug Dev Technol. 2014 ; 12 : 207 – 18 . Google Scholar Crossref Search ADS PubMed WorldCat 26. Haycock JW . 3D cell culture: A review of current approaches and techniques . Methods Mol Biol. 2011 ; 695 : 1 – 15 . Google Scholar Crossref Search ADS PubMed WorldCat 27. Legant WR , Pathak A, Yang MT et al. Microfabricated tissue gauges to measure and manipulate forces from 3D microtissues . Proc Natl Acad Sci U S A. 2009 ; 106 : 10097 – 102 . Google Scholar Crossref Search ADS PubMed WorldCat 28. Asmani M , Velumani S, Li Y et al. Fibrotic microtissue array to predict anti-fibrosis drug efficacy . Nat Commun. 2018 ; 9 :2066. OpenURL Placeholder Text WorldCat 29. Walker M , Rizzuto P, Godin M et al. Structural and mechanical remodeling of the cytoskeleton maintains tensional homeostasis in 3D microtissues under acute dynamic stretch . Sci Rep. 2020 ; 10 : 7696 . Google Scholar Crossref Search ADS PubMed WorldCat 30. Walker M , Godin M. Pelling AE. A vacuum-actuated microtissue stretcher for long-term exposure to oscillatory strain within a 3D matrix . Biomed Microdevices. 2018 ; 20 : 43 . Google Scholar Crossref Search ADS PubMed WorldCat 31. Vaughan MB , Howard EW, Tomasek JJ. Transforming growth factor-β1 promotes the morphological and functional differentiation of the myofibroblast . Exp Cell Res. 2000 ; 257 : 180 – 9 . Google Scholar Crossref Search ADS PubMed WorldCat 32. Zhao R , Chen CS, Reich DH. Force-driven evolution of mesoscale structure in engineered 3D microtissues and the modulation of tissue stiffening . Biomaterials. 2014 ; 35 : 5056 – 64 . Google Scholar Crossref Search ADS PubMed WorldCat 33. Zhao R , Boudou T, Wang W-G et al. Decoupling cell and matrix mechanics in engineered microtissues using magnetically actuated microcantilevers . Adv Mater. 2013 ; 25 : 1699 – 705 . Google Scholar Crossref Search ADS PubMed WorldCat 34. Lee SH , Dominguez R. Regulation of actin cytoskeleton dynamics in cells . Molecules and cells. 2010 ; 29 : 11 – 25 . OpenURL Placeholder Text WorldCat 35. Amano M , Ito M, Kimura K et al. Phosphorylation and activation of myosin by rho-associated kinase (rho- kinase) . J Biol Chem. 1996 ; 271 : 20246 – 9 . Google Scholar Crossref Search ADS PubMed WorldCat 36. Zhou H , Zhang KX, Li YJ et al. Fasudil hydrochloride hydrate, a rho-kinase inhibitor, suppresses high glucose-induced proliferation and collagen synthesis in rat cardiac fibroblasts . Clin Exp Pharmacol Physiol. 2011 ; 38 : 387 – 94 . Google Scholar Crossref Search ADS PubMed WorldCat 37. Fukushima M , Nakamuta M, Kohjima M et al. Fasudil hydrochloride hydrate, a rho-kinase (ROCK) inhibitor, suppresses collagen production and enhances collagenase activity in hepatic stellate cells . Liver Int. 2005 ; 25 : 829 – 38 . Google Scholar Crossref Search ADS PubMed WorldCat 38. Itoh Y , Kimoto K, Imaizumi M et al. Inhibition of RhoA/rho-kinase pathway suppresses the expression of type I collagen induced by TGF-β2 in human retinal pigment epithelial cells . Exp Eye Res. 2007 ; 84 : 464 – 72 . Google Scholar Crossref Search ADS PubMed WorldCat 39. Hinz B . The myofibroblast: Paradigm for a mechanically active cell . J Biomech. 2010 ; 43 : 146 – 55 . Google Scholar Crossref Search ADS PubMed WorldCat 40. Saums MK , Wang W, Han B et al. Mechanically and chemically tunable cell culture system for studying the myofibroblast phenotype . Langmuir. 2014 ; 30 : 5481 – 7 . Google Scholar Crossref Search ADS PubMed WorldCat 41. Booth AJ , Hadley R, Cornett AM et al. Acellular normal and fibrotic human lung matrices as a culture system for in vitro investigation . Am J Respir Crit Care Med. 2012 ; 186 : 866 – 76 . Google Scholar Crossref Search ADS PubMed WorldCat 42. Brown AC , Fiore VF, Sulchek TA et al. Physical and chemical microenvironmental cues orthogonally control the degree and duration of fibrosis-associated epithelial-to-mesenchymal transitions . J Pathol. 2013 ; 229 : 25 – 35 . Google Scholar Crossref Search ADS PubMed WorldCat 43. Liu F , Mih JD, Shea BS et al. Feedback amplification of fibrosis through matrix stiffening and COX-2 suppression . J Cell Biol. 2010 ; 190 : 693 – 706 . Google Scholar Crossref Search ADS PubMed WorldCat 44. Georges PC , Hui JJ, Gombos Z et al. Increased stiffness of the rat liver precedes matrix deposition: Implications for fibrosis . Am J Physiol - Gastrointest Liver Physiol. 2007 ; 293 : G1147 – 54 . Google Scholar Crossref Search ADS PubMed WorldCat 45. Perepelyuk M , Terajima M, Wang AY et al. Hepatic stellate cells and portal fibroblasts are the major cellular sources of collagens and lysyl oxidases in normal liver and early after injury . Am J Physiol - Gastrointest Liver Physiol. 2013 ; 304 : G605 – 14 . Google Scholar Crossref Search ADS PubMed WorldCat 46. Bond JE , Kokosis G, Ren L et al. Wound contraction is attenuated by fasudil inhibition of rho-associated kinase . Plast Reconstr Surg. 2011 ; 128 : 438e – 50e . Google Scholar Crossref Search ADS PubMed WorldCat 47. Ho TJ , Huang CC, Huang CY et al. Fasudil, a rho-kinase inhibitor, protects against excessive endurance exercise training-induced cardiac hypertrophy, apoptosis and fibrosis in rats . Eur J Appl Physiol. 2012 ; 112 : 2943 – 55 . Google Scholar Crossref Search ADS PubMed WorldCat 48. Huang X , Gai Y, Yang N et al. Relaxin regulates Myofibroblast contractility and protects against lung fibrosis . Am J Pathol. 2011 ; 179 : 2751 – 65 . Google Scholar Crossref Search ADS PubMed WorldCat 49. Goffin JM , Pittet P, Csucs G et al. Focal adhesion size controls tension-dependent recruitment of alpha-smooth muscle actin to stress fibers . J Cell Biol. 2006 ; 172 : 259 – 68 . Google Scholar Crossref Search ADS PubMed WorldCat 50. Hinz B . Masters and servants of the force: The role of matrix adhesions in myofibroblast force perception and transmission . European Journal of Cell Biology. 2006 ; 85 : 175 – 81 . Google Scholar Crossref Search ADS PubMed WorldCat 51. SHI Y , DONG Y, Duan Y et al. Substrate stiffness influences TGF-β1-induced differentiation of bronchial fibroblasts into myofibroblasts in airway remodeling . Mol Med Rep. 2013 ; 7 : 419 – 24 . Google Scholar Crossref Search ADS PubMed WorldCat 52. Huang X , Yang N, Fiore VF et al. Matrix stiffness-induced myofibroblast differentiation is mediated by intrinsic mechanotransduction . Am J Respir Cell Mol Biol. 2012 ; 47 : 340 – 8 . Google Scholar Crossref Search ADS PubMed WorldCat 53. Merryman WD , Lukoff HD, Long RA et al. Synergistic effects of cyclic tension and transforming growth factor-β1 on the aortic valve myofibroblast . Cardiovasc Pathol. 2007 ; 16 : 268 – 76 . Google Scholar Crossref Search ADS PubMed WorldCat 54. Gould RA , Chin K, Santisakultarm TP et al. Cyclic strain anisotropy regulates valvular interstitial cell phenotype and tissue remodeling in three-dimensional culture . Acta Biomater. 2012 ; 8 : 1710 – 9 . Google Scholar Crossref Search ADS PubMed WorldCat 55. Thomas Freeberg M , Thatcher TH, Sime PJ. Assessment of Myofibroblast phenotype with cellular stretch on substrates of different stiffness . American Journal of Respiratory and Critical Care Medicine. 2020 ; 201 : A2560 . OpenURL Placeholder Text WorldCat 56. Waxman AS , Kornreich BG, Gould RA et al. Interactions between TGFβ1 and cyclic strain in modulation of myofibroblastic differentiation of canine mitral valve interstitial cells in 3D culture . J Vet Cardiol. 2012 ; 14 : 211 – 21 . Google Scholar Crossref Search ADS PubMed WorldCat 57. Blaauboer ME , Smit TH, Hanemaaijer R et al. Cyclic mechanical stretch reduces myofibroblast differentiation of primary lung fibroblasts . Biochem Biophys Res Commun. 2011 ; 404 : 23 – 7 . Google Scholar Crossref Search ADS PubMed WorldCat 58. Desmouliere A , Redard M, Darby I et al. Apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar . Am J Pathol. 1995 ; 146 : 56 – 66 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 59. Kollmannsberger P , Bidan CM, Dunlop JWC et al. Tensile forces drive a reversible fibroblast-to-myofibroblast transition during tissue growth in engineered clefts . Sci Adv. 2018 ; 4 :eaao4881. OpenURL Placeholder Text WorldCat 60. Nagaraju CK , Robinson EL, Abdesselem M et al. Myofibroblast phenotype and reversibility of fibrosis in patients with end-stage heart failure . J Am Coll Cardiol. 2019 ; 73 : 2267 – 82 . Google Scholar Crossref Search ADS PubMed WorldCat 61. Hecker L , Jagirdar R, Jin T et al. Reversible differentiation of myofibroblasts by MyoD . Exp Cell Res. 2011 ; 317 : 1914 – 21 . Google Scholar Crossref Search ADS PubMed WorldCat 62. Kulkarni AB , Huh CG, Becker D et al. Transforming growth factor β1 null mutation in mice causes excessive inflammatory response and early death . Proc Natl Acad Sci U S A. 1993 ; 90 : 770 – 4 . Google Scholar Crossref Search ADS PubMed WorldCat 63. Blain EJ . Mechanical regulation of matrix metalloproteinases . Front Biosci. 2007 ; 12 : 507 – 27 . Google Scholar Crossref Search ADS PubMed WorldCat © The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Mechanical stretch sustains myofibroblast phenotype and function in microtissues through latent TGF-β1 activation
JF - Integrative Biology
DO - 10.1093/intbio/zyaa015
DA - 2020-09-07
UR - https://www.deepdyve.com/lp/oxford-university-press/mechanical-stretch-sustains-myofibroblast-phenotype-and-function-in-Lz8s7xvFCW
SP - 199
EP - 210
VL - 12
IS - 8
DP - DeepDyve
ER -