Function Of Substrate Stiffness Research Articles

Compliant substrates mitigate the senescence associated phenotype of stress induced mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are a promising cell population for musculoskeletal cell-based therapies due to their multipotent differentiation capacity and complex secretome. Cells from younger donors are mechanosensitive, evidenced by changes in cell morphology, adhesivity, and differentiation as a function of substrate stiffness in both two- and three-dimensional culture. However, MSCs from older individuals exhibit reduced differentiation potential and increased senescence, limiting their potential for autologous use. While substrate stiffness is known to modulate cell phenotype, the influence of the mechanical environment on senescent MSCs is poorly described. To address this question, we cultured irradiation induced premature senescent MSCs on polyacrylamide hydrogels and assessed expression of senescent markers, cell morphology, and secretion of inflammatory cytokines. Compared to cells on tissue culture plastic, senescent MSCs exhibited decreased markers of the senescence associated secretory phenotype (SASP) when cultured on 50 kPa gels, yet common markers of senescence (e.g., p21, CDKN2A, CDKN1A) were unaffected. These effects were muted in a physiologically relevant heterotypic mix of healthy and senescent MSCs. Conditioned media from senescent MSCs on compliant substrates increased osteoblast mineralization compared to conditioned media from cells on TCP. Mixed populations of senescent and healthy cells induced similar levels of osteoblast mineralization compared to healthy MSCs, further indicating an attenuation of the senescent phenotype in heterotypic populations. These data indicate that senescent MSCs exhibit a decrease in senescent phenotype when cultured on compliant substrates, which may be leveraged to improve autologous cell therapies for older donors.

Intercellular Adhesion Stiffness Moderates Cell Decoupling as a Function of Substrate Stiffness

The interplay between cell-cell and cell-substrate interactions is complex yet necessary for the formation and healthy functioning of tissues. The same mechanosensing mechanisms used by the cell to sense its extracellular matrix also play a role in intercellular interactions. We used the discrete element method to develop a computational model of a deformable cell that includes subcellular components responsible for mechanosensing. We modeled a three-dimensional cell pair on a patterned (two-dimensional) substrate, a simple laboratory setup to study intercellular interactions. We explicitly modeled focal adhesions and adherens junctions. These mechanosensing adhesions matured, becoming stabilized by force. We also modeled contractile stress fibers that bind the discrete adhesions. The mechanosensing fibers strengthened upon stalling. Traction exerted on the substrate was used to generate traction maps (along the cell-substrate interface). These simulated maps are compared to experimental maps obtained via traction force microscopy. The model recreates the dependence on substrate stiffness of the tractions’ spatial distribution, contractile moment of the cell pair, intercellular force, and number of focal adhesions. It also recreates the phenomenon of cell decoupling, in which cells exert forces separately when substrate stiffness increases. More importantly, the model provides viable molecular explanations for decoupling: mechanosensing mechanisms are responsible for competition between different fiber-adhesion configurations present in the cell pair. The point at which an increasing substrate stiffness becomes as high as that of the cell-cell interface is the tipping point at which configurations that favor cell-substrate adhesion dominate over those favoring cell-cell adhesion. This competition is responsible for decoupling.

Integrin Binding Dynamics Modulate Ligand-Specific Mechanosensing in Mammary Gland Fibroblasts.

SummaryThe link between integrin activity regulation and cellular mechanosensing of tissue rigidity, especially on different extracellular matrix ligands, remains poorly understood. Here, we find that primary mouse mammary gland stromal fibroblasts (MSFs) are able to spread efficiently, generate high forces, and display nuclear YAP on soft collagen-coated substrates, resembling the soft mammary gland tissue. We describe that loss of the integrin inhibitor, SHARPIN, impedes MSF spreading specifically on soft type I collagen but not on fibronectin. Through quantitative experiments and computational modeling, we find that SHARPIN-deficient MSFs display faster force-induced unbinding of adhesions from collagen-coated beads. Faster unbinding, in turn, impairs force transmission in these cells, particularly, at the stiffness optimum observed for wild-type cells. Mechanistically, we link the impaired mechanotransduction of SHARPIN-deficient cells on collagen to reduced levels of collagen-binding integrin α11β1. Thus integrin activity regulation and α11β1 play a role in collagen-specific mechanosensing in MSFs.

PO-268 The interaction of hERG1 potassium channels with integrin receptors perturbs the force transduction machinery in pancreatic cancer

IntroductionIon channels regulate cell proliferation, differentiation, and migration through cell-cell and cell–extracellular matrix interactions mediated by integrins. K+ flux through the human ether-à-gogo–related gene 1 (hERG1) channel shapes action potential firing in excitable cells, e.g. cardiomyocytes. Its abundance is often aberrantly high in human tumours, including the pancreatic ductal adenocarcinoma (PDAC). We recently demonstrated that the direct interaction of β1 integrins with hERG1 channels in cancer cells stimulated distinct signalling pathways that depended on the conformational state of hERG1 and affected different aspects of tumour progression.1 We hypothesised that hERG1 channels compromise the PDAC mechano-reciprocity, the ability to dynamically respond to externally applied forces by exerting forces, which enhances invasion and compromises treatment.Material and methodsUsing elastic micropillar arrays of varying stiffness,2 we quantified the β1-integrin mediated forces exerted by PANC-1 cells. Micropillars coating with collagen and/or fibronectin derived peptides allowed us to direct cell surface receptor binding specificity (i.e. α2β1 and α5β1 integrins).Results and discussionsIndependently of the substrate coating, PANC-1 cells exerted higher forces as a function of substrate stiffness, as already demonstrated for other cell types. Remarkably, the disruption of the β1/hERG1 interaction through E4031 inhibition of hERG1 channels resulted in a significant increase in the detected cellular forces. Our results suggest that in addition to alter distinct signalling pathways1 the direct interaction of β1 integrins and hERG1 channels perturbs the force transduction machinery.ConclusionThese findings encouraged us to develop bispecific antibodies (scDb, single-chain diabody) binding to the β1/hERG1 complex that are now being validated in PDAC cell lines. In particular, we have developed a bispecific antibody (scDb-β1/hERG1),3 which is composed by the variable domains (VH and VL chains) of monoclonal antibodies binding two different antigens, hERG1 and β1 integrin.ReferencesBecchetti, et al. Science Signaling2017;10, eaaf3236.van Hoorn, et al. Nano Lett2014;14:4257.A. Arcangeli, C. Duranti, S. Crescioli, L. Carraresi. Patent Ref: 102017000083637 (University of Florence).

Abstract 183: HERG1 potassium channels perturb the β1 integrins mediated force transduction machinery in pancreatic cancer

Abstract Ion channels regulate cell proliferation, differentiation, and migration in normal and neoplastic cells through cell-cell and cell-extracellular matrix (ECM) transmembrane receptors called integrins. K+ flux through the human ether-à-gogo-related gene 1 (hERG1) channel shapes action potential firing in excitable cells such as cardiomyocytes. Its abundance is often aberrantly high in human tumors, including the pancreatic ductal adenocarcinoma (PDAC). We recently demonstrated that the direct interaction of β1 integrins with hERG1 channels in cancer cells stimulated distinct signaling pathways that depended on the conformational state of hERG1 and affected different aspects of tumor progression [1]. We hypothesized that hERG1 channels compromise the PDAC mechano-reciprocity, the ability to dynamically respond to externally applied forces by exerting forces, which enhances invasion and compromises treatment. Using elastic micropillar arrays of varying stiffness (20-150 kPa) [2], we quantified the β1-integrin mediated forces exerted by PANC-1 cells. Micropillars coating with collagen and/or fibronectin derived peptides allowed us to direct cell surface receptor binding specificity (i.e. α2β1 and α5β1 integrins). Independently of the substrate coating, PANC-1 cells exerted higher forces as a function of substrate stiffness, as already demonstrated for other cell types. Remarkably, the disruption of the β1/hERG1 interaction through E4031 inhibition of hERG1 channels resulted in a significant increase in the detected cellular forces by ~20%, accompanied by an increase of focal adhesion areas as demonstrated by staining with vinculin. Our results suggest that, in addition to alter distinct signaling pathways and tumor progression [1], the direct interaction of β1 integrins and hERG1 channels perturbs the force transduction machinery. These findings encouraged us to develop bispecific antibodies (scDb, single-chain diabody) binding to the β1/hERG1 complex that are now being validated in PDAC cell lines. In particular, we have developed a bispecific antibody (scDb-β1/hERG1) [3], which is composed by the variable domains (VH and VL chains) of monoclonal antibodies binding two different antigens, hERG1 and β1 integrin. Data obtained from in vitro studies demonstrates that the scDb antibody impacts cell viability and migratory behavior in Mia PaCa-2 cell lines, holding promises for translational impact after a further validation in vivo as a novel candidate in cancer therapeutics. [1] Becchetti et al. Science Signaling (2017), 10 (473), eaaf3236. [2] van Hoorn et al. Nano Lett (2014), 14, 4257-4262. [3] Patent: “Novel antibodies”. Inventors: Annarosa Arcangeli, Claudia Duranti, Silvia Crescioli, Laura Carraresi. Patent Deposit: July 2017. Patent Ref: 102017000083637 (University of Florence). Citation Format: Stefano Coppola, Claudia Duranti, Annarosa Arcangeli, Thomas Schmidt. HERG1 potassium channels perturb the β1 integrins mediated force transduction machinery in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 183.

CELL‐MATRIX INTERACTIONS AND MECHANOSENSITIVE PATHWAYS IN UTERINE LEIOMYOMA (FIBROID) PROLIFERATION

BackgroundUterine leiomyomas (fibroids; UF) are benign myometrial neoplasms that produce clinically‐relevant symptoms in >25% of all women including excessive menstrual bleeding and infertility, and represent the leading cause of hysterectomy (~600,000/year in the US). Despite their importance, the mechanisms that promote UF growth are still not well‐understood. UFs are comprised of smooth muscle cells and fibroblasts with substantial extracellular matrix (ECM). While cells within UFs produce ECM, the role of ECM per se in modulating cell properties towards overall tumor growth is not known. This study tests the hypothesis that UF cells are mechanically sensitive, and that the ECM provides a positive feedback mechanism which further promotes UF growth.MethodsCells were enzymatically isolated from UFs of patients undergoing myomectomy at Mayo Clinic Rochester (IRB approved). Fibroid cells were plated onto soft (0.2kPa) vs. stiff (64kPa) polydimethylsiloxane (PDMS) elastomer substrates or standard tissue culture plastic substrates (~100GPa) for 24 h in DMEM containing 10% FBS prior to RNA isolation and qPCR analysis. Transcript levels for known ECM and profibrotic genes were determined. Nuclear localization of YAP and TAZ was visualized using immunofluorescence and quantified using a Cytation5 imaging system. Separately, 2‐color fluorescence immunohistochemistry for YAP and TAZ was performed on formalin fixed; paraffin embedded normal myometrium and fibroid tissue sections. Transciption level and nuclear localization calculations were performed with Kruskall‐Wallis and Wilcoxon Sign‐Rank tests, respectively.ResultsSignificant increases in transcripts for ECM gene Collagen 1 (COL1A1) were noted for UF cells plated on stiff PDMS and standard plastic compared to soft PDMS. The pro‐fibrotic genes Connective Tissue Growth Factor (CTGF), alpha smooth muscle actin (ACTA2), Endothelin 1 (EDN1), and plasminogen activator inhibitor 1 (SERPINE1) were progressively increased in expression going from soft PDMS substrate to plastic. The anti‐fibrotic gene, cyclooxygenase 2 (PTGS2) was decreased on stiff PDMS and plastic compared to soft PDMS. In cells plated on plastic, there was a significant decrease in YAP/TAZ nuclear localization in cells treated with the Rho‐kinase inhibitor Y‐27632, consistent with a mechanobiologic feedback. Immunohistochemistry showed a two‐fold increase in YAP/TAZ nuclear localization for fibroid tissue compared to normal myometrium.ConclusionsBased on the growing appreciation that ECM mechanical properties contribute to UF pathobiology and fibrotic matrix deposition, we show enhanced ECM and profibrotic gene expression by fibroid cells as a function of substrate stiffness. These results demonstrate a feedback mechanism by which UFs progress through matrix stiffness‐mediated ECM deposition. Here, the mechanosensitive pathways of YAP and TAZ may be involved in transducing ECM signals to UF growth.Support or Funding InformationNoneThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

From Adhesion to Wetting: Contact Mechanics at the Surfaces of Super-Soft Brush-Like Elastomers

Fundamental understanding of rigid particle indentation into soft elastic substrates has been elusive for decades. In conventional heterogeneous and multicomponent systems, the ill-defined interplay between elastic and capillary forces has confounded explanation of the crossover region between the classical wetting and adhesion regimes. Herein, we study the indentation behavior of micrometer-sized silica particles on supersoft, solvent-free PDMS elastomers with brush-like network strands. By varying the side chain grafting density and the cross-linking density of the networks, we control their elastic modulus from ∼1 to 100 kPa without adding solvent. This isostructurally regulated balance between elastic and capillary forces allows for accurate mapping of the entire range of particle–substrate interactions by measuring indentation depth as a function of substrate stiffness and particle radius. A generalized theoretical model, accounting for the collaborative contribution of both forces to the system free...

Mechanosensing of substrate stiffness regulates focal adhesions dynamics in cell

Cell mechanical recognition of extracellular matrix determines the cell activities and functions. Focal adhesions are part of the cell mechanosensing machinery and, operating at the very dynamic interface between cell and extracellular matrix, can operate this recognition and trigger conformational, functional and behavioral modification of the cell. To investigate how the dynamic of assembly and disassembly of focal adhesion are influenced by the substrate mechanics we developed a novel procedure. The analysis consists of the over time tracking of focal adhesion structures in a stable cell line of NIH/3T3 expressing fluorescent pmKate2-paxillin. From collected signals and by their autocorrelation we evaluated the average lifetime and assembly rate of focal adhesion as function of substrate stiffness. Further, by signals cross-correlation we obtained information about the mechanical nature of cytoskeleton and its network. This quantitative approach to focal adhesion dynamics characterization was presented in this study as an investigation tool for cell mechanobiology.

Extracellular Matrix Stiffness Controls VEGF Signaling and Processing in Endothelial Cells.

Vascular endothelial growth factor A (VEGF) drives endothelial cell maintenance and angiogenesis. Endothelial cell behavior is altered by the stiffness of the substrate the cells are attached to suggesting that VEGF activity might be influenced by the mechanical cellular environment. We hypothesized that extracellular matrix (ECM) stiffness modifies VEGF-cell-matrix tethering leading to altered VEGF processing and signaling. We analyzed VEGF binding, internalization, and signaling as a function of substrate stiffness in endothelial cells cultured on fibronectin (Fn) linked polyacrylamide gels. Cell produced extracellular matrices on the softest substrates were least capable of binding VEGF, but the cells exhibited enhanced VEGF internalization and signaling compared to cells on all other substrates. Inhibiting VEGF-matrix binding with sucrose octasulfate decreased cell-internalization of VEGF and, inversely, heparin pre-treatment to enhance Fn-matrix binding of VEGF increased cell-internalization of VEGF regardless of matrix stiffness. β1 integrins, which connect cells to Fn, modulated VEGF uptake in a stiffness dependent fashion. Cells on hard surfaces showed decreased levels of activated β1 and inhibition of β1 integrin resulted in a greater proportional decrease in VEGF internalization than in cells on softer matrices. Extracellular matrix binding is necessary for VEGF internalization. Stiffness modifies the coordinated actions of VEGF-matrix binding and β1 integrin binding/activation, which together are critical for VEGF internalization. This study provides insight into how the microenvironment may influence tissue regeneration and response to injury and disease. J. Cell. Physiol. 231: 2026-2039, 2016. © 2016 Wiley Periodicals, Inc.

Adaptative Response of Cell Cytoskeleton Rheology and Ordering Governs Matrix Rigidity Sensing

Matrix rigidity sensing regulates a large variety of cellular processes and has important implications for tissue development and disease. However, how cells probe matrix rigidity, and hence respond to it, remains unclear. Here, we show that rigidity sensing and adaptation emerge naturally from actin cytoskeleton remodelling. We report that cells markedly modify their internal rheology and traction forces in response to substrate stiffness. Our in vitro experiments and theoretical modeling demonstrate a bi-phasic behavior of the actin cytoskeleton, which transitions sharply from fluid on soft substrates to solid on stiffer ones. Furthermore, we find that increasing substrate stiffness correlates with the emergence of orientational order in actin stress fibers, which exhibit an isotropic to nematic transition that we characterize quantitatively in the framework of active matter theory6. This finding implicates mechanisms mediated not only by the activation of local adhesion complexes, as it is usually assumed, but also by a large-scale reinforcement of actin structures under stress. As such, this unanticipated rheological response of the cell represents the mechanical driver of cell shape changes and cell polarity as a function of substrate stiffness.

Mechanobiology of mRNA Localization in Breast Cancer Cells

Introduction: Metastasis occurs when cancer cells form secondary tumors in distant areas of the body. Localization of RNAs in lamellipodial regions has been proposed to play an important role during metastatic progression. In one pathway, the tumor suppressor protein, adenomatous polyposis coli (APC) targets RNAs to cell protrusions. APC -associated mRNAs have been implicated in cellular migration and metastatic progression. Therefore, localization or not of these mRNAs has functional significance in cellular migration and metastasis. Additionally, It has been shown that cancer cells modulate their gene expression in response to the mechanical properties of the substrate. Furthermore, mRNA localization at adhesion sites is influenced by mechanical tension, which is adjusted by cells as a function of the mechanical properties of the cell environment. Therefore, mechanical properties of tissues may play a role during metastasis by modulating localization of mRNAs. As a result, this study investigates APC- associated mRNA localization as a function of substrate stiffness. Methods: We used the MCF10A cell series, a breast cancer progression model composed of cell lines representing pre-malignant to invasive transformation to investigate mRNA localization. By using in situ hybridization to fluorescently label mRNAs, and micropatterned polyacrylamide gels of varying stiffness, we observed APC associated mRNA localization. Glu-tubulin and vinculin were immunostained to study their relationship to mRNA localization. Results: On stiffer substrates (280kPa), we observed increased mRNA localization compared to softer substrates (0.87kPa). Staining of cytoskeletal elements such as Glu-tubulin and vinculin showed a correlation between the abundance and location of the proteins and APC-associated mRNAs. These results reveal biophysical roles of adhesion and cytoskeletal tension in regulating mRNAs to protrusions.

Influence of the PDMS substrate stiffness on the adhesion of Acanthamoeba castellanii.

Background: Mechanosensing of cells, particularly the cellular response to substrates with different elastic properties, has been discovered in recent years, but almost exclusively in mammalian cells. Much less attention has been paid to mechanosensing in other cell systems, such as in eukaryotic human pathogens.Results: We report here on the influence of substrate stiffness on the adhesion of the human pathogen Acanthamoebae castellanii (A. castellanii). By comparing the cell adhesion area of A. castellanii trophozoites on polydimethylsiloxane (PDMS) substrates with different Young’s moduli (4 kPa, 29 kPa, and 128 kPa), we find significant differences in cell adhesion area as a function of substrate stiffness. In particular, the cell adhesion area of A. castellanii increases with a decreasing Young’s modulus of the substrate.Conclusion: The dependence of A. castellanii adhesion on the elastic properties of the substrate is the first study suggesting a mechanosensory effect for a eukaryotic human pathogen. Interestingly, the main targets of A. castellanii infections in the human body are the eye and the brain, i.e., very soft environments. Thus, our study provides first hints towards the relevance of mechanical aspects for the pathogenicity of eukaryotic parasites.

Cell Viscoelasticity as a Function of Substrate Stiffness

While it is known that cells respond to the mechanical properties of their environment, limited information is available regarding mechanosensing and the means by which such stimuli are transduced. The cytoskeleton, a critical component used by cells to sense substrate stiffness, determines the viscoelasticity of cells. Information on cell viscoelasticity as a function of substrate stiffness and vimentin levels can lead to a larger composite model of the mechanism through which cells respond to external mechanical stimuli.3T3 fibroblasts were grown on collagen-coated polyacrylamide gels, of which the stiffness was controlled by varying both the concentrations of acrylamide and bisacrylamide. An atomic force microscope (AFM) was used to perform dynamic micromechanical tests. The force-mapping mode was employed to obtain 24 by 24 maps of force curves. For each force curve, the AFM first indents a cell with a 700 pN force and then applies a 10 Hz sinusoidal strain to the cell. The storage, E', and loss modulus, E'', were calculated by fitting the sinusoidal portion of the applied force and resulting indentation to obtain the amplitude and phase offset.Both E' and E'' increase with substrate stiffness. While the increase in E' is consistent with literature, we found that the ratio of E'' to E' decreases as substrate stiffness increases. This agrees with our hypothesis that cells become more solid-like as the elastic modulus of the extracellular matrix increases. Additionally, the value of E' was higher in vimentin null cells, suggesting that cells change their cytoskeletal composition to adapt to the loss of vimentin. This may involve an increase in the concentration of actin filaments, which contribute more to the elasticity than vimentin. Further experiments will investigate the potential link between vimentin expression and the mechanosensing ability of the cells.

Optimality of Force Transmission in a Motor-Clutch Cellular Adhesion Model

Microenvironmental mechanics play an important, but variable, role in determining cell morphology, traction, migration, proliferation, and differentiation with potential impacts on tumor development, growth, and invasion. Interestingly, some cell types have shown increasing migration and traction force as a function of substrate stiffness, while others have shown decreasing migration and traction force. These seemingly contradictory results may be explained by a motor-clutch model of cellular adhesion and force transmission which exhibits a maximum in traction force with respect to stiffness and may be tuned to different stiffness optima. Both stochastic and deterministic castings of the motor-clutch model provide a basis to explain the tuning of cells to different microenvironmental mechanics. A sensitivity analysis of the stochastic model suggests that molecular motors and adhesion clutches must approximately balance each other to achieve stiffness sensitivity. Consequently, individual parameters changes, which favor only the motors or the clutches, have little effect in shifting the stiffness optimum because the system loses stiffness sensitivity altogether. However, dual parameters changes, such that motors and clutches remain balanced, can shift the stiffness optimum over several orders of magnitude. This optimum occurs on the stiffness at which the time for all clutches to bind equals the cycle time of adhesion load and fail. At stiffnesses above this optimum, fewer than the maximum clutches bind, so the clutches are not utilized to their fullest extent. At stiffnesses below the optimum, clutches spontaneously fail at low loads because of the long cycle time, again resulting in an inefficient use of clutches. This determinant of the optimum stiffness was applied in conjunction with the deterministic motor-clutch model to derive a dimensionless quantity defining model behavior at any particular stiffness.

Determinants of Maximal Force Transmission in a Motor-Clutch Model of Cell Traction in a Compliant Microenvironment

The mechanical stiffness of a cell’s environment exerts a strong, but variable, influence on cell behavior and fate. For example, different cell types cultured on compliant substrates have opposite trends of cell migration and traction as a function of substrate stiffness. Here, we describe how a motor-clutch model of cell traction, which exhibits a maximum in traction force with respect to substrate stiffness, may provide a mechanistic basis for understanding how cells are tuned to sense the stiffness of specific microenvironments. We find that the optimal stiffness is generally more sensitive to clutch parameters than to motor parameters, but that single parameter changes are generally only effective over a small range of values. By contrast, dual parameter changes, such as coordinately increasing the numbers of both motors and clutches offer a larger dynamic range for tuning the optimum. The model exhibits distinct regimes: at high substrate stiffness, clutches quickly build force and fail (so-called frictional slippage), whereas at low substrate stiffness, clutches fail spontaneously before the motors can load the substrate appreciably (a second regime of frictional slippage). Between the two extremes, we find the maximum traction force, which occurs when the substrate load-and-fail cycle time equals the expected time for all clutches to bind. At this stiffness, clutches are used to their fullest extent, and motors are therefore resisted to their fullest extent. The analysis suggests that coordinate parameter shifts, such as increasing the numbers of motors and clutches, could underlie tumor progression and collective cell migration.

Control of VEGF‐Endothelial Response by ECM Stiffness

Vascular endothelial growth factor (VEGF) drives both endothelial cell maintenance and normal/pathological angiogenesis. Recent research has suggested that mechanical signaling due to the stiffness of the cell contact surface may also have profound effects on endothelial cell behavior. To study VEGF signaling as a function of substrate stiffness, endothelial cells were cultured on fibronectin linked polyacrylamide gels and treated with VEGF. To analyze the response to VEGF we developed a custom MATLAB software script to analyze calcium flux movies. The script is able to individually identify cells, normalize each calcium signal value to the cell's individual background, and analyze the data. A variety of outputs are produced such as the magnitude of response, number of local maximums each cell experiences and clusters of coordinated cells. Utilizing this tool, we observed that VEGF‐stimulated intracellular calcium signaling varies in both magnitude and signaling kinetics depending on the stiffness of the substrate. At low VEGF concentrations we found maximal response (2–3.5 fold increases) at moderate stiffness whereas at high concentrations of VEGF the softest substrates showed the most intense response, but the response dissipated the fastest. We show that extracellular matrix stiffness can differentially drive endothelial response to VEGF. Supported by HL088572 and AHAF M2012014.

Investigating the role of substrate stiffness in the persistence of valvular interstitial cell activation.

During heart valve remodeling and in many disease states, valvular interstitial cells (VICs) shift to an activated myofibroblast phenotype characterized by enhanced synthetic and contractile activity. Pronounced alpha smooth muscle actin (αSMA)-positive stress fibers, the hallmark of activated myofibroblasts, are also observed in VICs cultured on stiff substrates especially in the presence of transforming growth factor-beta1 (TGF-β1), however, the detailed relationship between stiffness and VIC phenotype has not been explored. The goal of this study was to characterize VIC activation as a function of substrate stiffness over a wide range of stiffness levels including that of diseased valves (stiff), normal valves (compliant), and hydrogels for heart valve tissue engineering (very soft). VICs obtained from porcine aortic valves were cultured on stiff tissue culture plastic to activate them, then, cultured on collagen-coated polyacrylamide substrates of predefined stiffness in a high-throughput culture system to assess the persistence of activation. Metrics extracted from regression analysis demonstrate that relative to a compliant substrate, stiff substrates result in higher cell numbers, more pronounced expression of αSMA-positive stress fibers, and larger spread area which is in qualitative agreement with previous studies. Our data also indicate that VICs require a much lower substrate stiffness level to "deactivate" them than previously thought. The high sensitivity of VICs to substrate stiffness demonstrates the importance of the mechanical properties of materials used for valve repair or for engineering valve tissue.

Stress Fiber Organization and Dynamics in Cells Adhered to Substrates of Varying Stiffness

AbstractCellular morphology, locomotion and division are closely related to the stiffness of the substrates onto which the cells are cultured. The stiffness of cultured cells typically increases with the stiffness of the substrate. This increase correlates with increased contractility and the development of a meshwork of parallel and cross-linked stress fibers along the contacting surface. Many questions remain regarding the mechanisms that underlie cell-level cytoskeletal remodeling during mechanosensing. To better quantify the morphology and dynamics of the actomyosin cytoskeleton as a function of substrate stiffness, we used confocal microscopy to image cultured HeLa cells that stably express myosin regulatory light chain tagged with GFP (MRLC-GFP). We cultured cells on poly-acrylamide substrates coated with collagen I, with stiffness ranging from 0.4 kPa to 60 kPa. We used image segmentation methods to measure stress fiber and cortical myosin distributions in static 3D images. Time-lapse recordings show a connected stress fiber meshwork that evolves through new stress fiber formation on the cell periphery, contractile activity, and stress fiber merging and splitting over times of order hours. Quantitative analysis is suggestive of kinetic models that explore how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness.

Mitochondrial Trafficking on Axons as a Function of Substrate Stiffness

Mitochondria are the energy machinery necessary for the maintenance of axonal growth, calcium management and other neuronal functions and disruption of their transport results in neurological disease. Due to the length of neurite extensions, mitochondria travel along microtubules and actin filaments to reach different places along the axon; thus, their transport and density is regulated by the activity of axonal zones and growth cones. Mitochondrial trafficking has been linked to axonal growth and thus is an important property in the study of neuroregeneration. It has previously been shown that axonal growth depends on substrate stiffness, suggesting that the mechanical properties of the substrate might play an important role in axonal transport. In this work we analyzed the transport of mitochondria along the axon as well as the distribution in the growth cone as a function of substrate stiffness, by imaging axonal transport of mitochondria in vitro on cultured rodent DRGs. These results provide insight into the transport mechanisms along the axon as a function of substrate stiffness. Furthermore, these results deepen our knowledge of the effects of mechanical properties of the substrate on the activity of axons and growth cones and show that mitochondrial trafficking may play a role in these effects.

Leukocyte Transmigration is Mediated by Endothelial Cell Contractile Forces and Substrate Stiffness

Leukocyte transmigration through the vascular endothelium is a crucial step in the normal immune response. However, in the cardiovascular disease (CVD) of atherosclerosis, an excess of leukocytes adhere to and transmigrate through the endothelium, and progression of this disease is associated with arterial stiffening and variance in mechanical force transduction. In this study we investigated the mechanics of leukocyte transmigration in an in vitro model of the vascular endothelium. We modeled healthy versus diseased blood vessels through manipulation of substrate stiffness using polyacrylamide gels, coated with extracellular matrix protein and plated with human umbilical vein endothelial cell (HUVEC) monolayers. The HUVEC monolayers were activated with tumor necrosis factor-alpha to mimic inflammatory conditions. We observed that leukocyte transmigration through HUVEC monolayers increases with stiffness below the endothelium, and we hypothesized that substrate stiffness changes the biophysical properties of the endothelium to produce this effect. Using an array of biophysical techniques, we first evaluated the adhesion protein expression, stiffness, morphology, cytoskeletal arrangement, and cell-substrate adhesion of HUVEC monolayers as a function of substrate stiffness; however, none of these properties could account for the transmigration behavior. We also explored the role of endothelial cell-cell adhesion and myosin light chain kinase (MLCK)-dependent endothelial cell contraction. We observed that (1) decreasing cell-cell adhesion increases transmigration on soft substrates and (2) inhibition of MLCK and endothelial cell contraction normalizes the effects of substrate stiffness by reducing leukocyte transmigration on stiff substrates without affecting transmigration on soft substrates. These results provide strong evidence that neutrophil transmigration is regulated by MLCK-mediated generation of gaps at cell borders through endothelial cell contractile forces which depend on substrate stiffness.