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Christopher Chen highlights the early studies of mechanoregulation of cell–matrix adhesions that established mechanobiology as a cross-discplinary research field

Dendritic cell migration to the T-cell-rich areas of the lymph node is essential for their ability to initiate the adaptive immune response. While it has been shown that the actin cytoskeleton is required for normal DC migration, the role of many of the individual cytoskeletal molecules is poorly understood. In this study, we investigated the contribution of the Arp2/3 complex binding protein, haematopoietic lineage cell-specific protein 1 (HS1), to DC migration and force generation. We quantified the random migration of HS1−/− DCs on 2D micro-contact printed surfaces and found that in the absence of HS1, DCs have greatly reduced motility and speed. This same reduction in motility was recapitulated when adding Arp2/3 complex inhibitor to WT DCs or using DCs deficient in WASP, an activator of Arp2/3 complex-dependent actin polymerization. We further investigated the importance of HS1 by measuring the traction forces of HS1−/− DCs on micropost array detectors (mPADs). In HS1 deficient DCs, there was a significant reduction in force generation (3.96 ± 0.40 nN per cell) compared to WT DCs (13.76 ± 0.84 nN per cell). Interestingly, the forces generated in DCs lacking WASP were only slightly reduced compared to WT DCs. Taken together, these findings show that HS1 and Arp2/3 complex-mediated actin polymerization are essential for the most efficient DC random migration and force generation.

The integrity of the endothelial barrier between circulating blood and tissue is important for blood vessel function and, ultimately, for organ homeostasis. Here, we developed a vessel-on-a-chip with perfused endothelialized channels lined with human bone marrow stromal cells, which adopt a mural cell-like phenotype that recapitulates barrier function of the vasculature. In this model, barrier function is compromised upon exposure to inflammatory factors such as LPS, thrombin, and TNFα, as has been observed in vivo. Interestingly, we observed a rapid physical withdrawal of mural cells from the endothelium that was accompanied by an inhibition of endogenous Rac1 activity and increase in RhoA activity in the mural cells themselves upon inflammation. Using a system to chemically induce activity in exogenously expressed Rac1 or RhoA within minutes of stimulation, we demonstrated RhoA activation induced loss of mural cell coverage on the endothelium and reduced endothelial barrier function, and this effect was abrogated when Rac1 was simultaneously activated. We further showed that

In spite of the vast collective experience in tissue engineering, control of both tissue architecture and scale are fundamental translational roadblocks. An experimental framework that enables investigation into how architecture and scaling may be coupled is needed. Here, we introduce an approach called ‘SEEDs’ (‘in Situ Expansion of Engineered Devices’), in which we fabricate a structurally organized engineered tissue unit that expands in response to regenerative cues after implantation. We find that tissues containing pre-patterned human primary hepatocytes, endothelial cells, and stromal cells in degradable hydrogel expand over 50-fold over the course of 11 weeks in animals with liver injury, with concomitant increased function as characterized by the production of multiple human liver proteins. Histologically, we observe the emergence of stereotypical microstructure via coordinated growth of hepatocytes in close juxtaposition with a perfused, chimeric vasculature. Importantly, we demonstrate the utility of this platform for probing the impact of multicellular geometric architecture on tissue expansion in response to regenerative cues. This approach represents a hybrid strategy that harnesses both biology and engineering to deploy a limited cell mass more efficiently than either approach could do in isolation, and thus offers a new convergent paradigm for tissue engineering.

SummarySwitch-like activation of the Spindle Assembly Checkpoint (SAC) is critical for accurate chromosome segregation during cell division. To determine the mechanisms that implement it, we engineered an ectopic, kinetochore-independent SAC activator, the “eSAC”. The eSAC stimulates the SAC signaling cascade by artificially dimerizing the Mps1 kinase domain and a cytosolic KNL1 phosphodomain, the signaling scaffold in the kinetochore. Quantitative analyses and mathematical modeling of the eSAC reveal that the recruitment of multiple SAC proteins by the KNL1 phosphodomain stimulates synergistic signaling, which enables a small number of KNL1 molecules produce a disproportionately strong anaphase-inhibitory signal. However, when multiple KNL1 molecules signal concurrently, they compete for a limited cellular pool of SAC proteins. This frustrates synergistic signaling and modulates signal output. Together, these mechanisms institute automatic gain control – inverse, non-linear scaling between the signal output per kinetochore and the unattached kinetochore number, and thus enact the SAC switch.

We describe a multiscale model that incorporates force-dependent mechanical plasticity induced by interfiber cross-link breakage and stiffness-dependent cellular contractility to predict focal adhesion (FA) growth and mechanosensing in fibrous extracellular matrices (ECMs). The model predicts that FA size depends on both the stiffness of ECM and the density of ligands available to form adhesions. Although these two quantities are independent in commonly used hydrogels, contractile cells break cross-links in soft fibrous matrices leading to recruitment of fibers, which increases the ligand density in the vicinity of cells. Consequently, although the size of focal adhesions increases with ECM stiffness in nonfibrous and elastic hydrogels, plasticity of fibrous networks leads to a departure from the well-described positive correlation between stiffness and FA size. We predict a phase diagram that describes nonmonotonic behavior of FA in the space spanned by ECM stiffness and recruitment index, which describes the ability of cells to break cross-links and recruit fibers. The predicted decrease in FA size with increasing ECM stiffness is in excellent agreement with recent observations of cell spreading on electrospun fiber networks with tunable cross-link strengths and mechanics. Our model provides a framework to analyze cell mechanosensing in nonlinear and inelastic ECMs.

During development, cells undergo dramatic changes in their morphology. By affecting contact geometry, these morphological changes could influence cellular communication. However, it has remained unclear whether and how signaling depends on contact geometry. This question is particularly relevant for Notch signaling, which coordinates neighboring cell fates through direct cell-cell signaling. Using micropatterning with a receptor trans-endocytosis assay, we show that signaling between pairs of cells correlates with their contact area. This relationship extends across contact diameters ranging from micrometers to tens of micrometers. Mathematical modeling predicts that dependence of signaling on contact area can bias cellular differentiation in Notch-mediated lateral inhibition processes, such that smaller cells are more likely to differentiate into signal-producing cells. Consistent with this prediction, analysis of developing chick inner ear revealed that ligand-producing hair cell precursors have smaller apical footprints than non-hair cells. Together, these results highlight the influence of cell morphology on fate determination processes.

A major challenge in tissue engineering is the development of materials that can support angiogenesis, wherein endothelial cells from existing vasculature invade the surrounding matrix to form new vascular structures. To identify material properties that impact angiogenesis, here we have developed an in vitro model whereby molded tubular channels inside a synthetic hydrogel are seeded with endothelial cells and subjected to chemokine gradients within a microfluidic device. To accomplish precision molding of hydrogels and successful integration with microfluidics, we developed a class of hydrogels that could be macromolded and micromolded with high shape and size fidelity by eliminating swelling after polymerization. Using this material, we demonstrate that matrix degradability switches three-dimensional endothelial cell invasion between two distinct modes: single-cell migration and the multicellular, strand-like invasion required for angiogenesis. The ability to incorporate these tunable hydrogels into geometrically constrained settings will enable a wide range of previously inaccessible biomedical applications., The fabrication of vascularized 3D tissues requires an understanding of how material properties govern endothelial cell invasion into the surrounding matrix. Here the authors integrate a non-swelling synthetic hydrogel with a microfluidic device to study chemokine gradient-driven angiogenic sprouting and find that matrix degradability modulates the collectivity of cell migration.

Ischemia-reperfusion injury (IRI) is a leading cause of AKI. This common clinical complication lacks effective therapies and can lead to the development of CKD. The αvβ5 integrin may have an important role in acute injury, including septic shock and acute lung injury. To examine its function in AKI, we utilized a specific function-blocking antibody to inhibit αvβ5 in a rat model of renal IRI. Pretreatment with this anti-αvβ5 antibody significantly reduced serum creatinine levels, diminished renal damage detected by histopathologic evaluation, and decreased levels of injury biomarkers. Notably, therapeutic treatment with the αvβ5 antibody 8 hours after IRI also provided protection from injury. Global gene expression profiling of post-ischemic kidneys showed that αvβ5 inhibition affected established injury markers and induced pathway alterations previously shown to be protective. Intravital imaging of post-ischemic kidneys revealed reduced vascular leak with αvβ5 antibody treatment. Immunostaining for αvβ5 in the kidney detected evident expression in perivascular cells, with negligible expression in the endothelium. Studies in a three-dimensional microfluidics system identified a pericyte-dependent role for αvβ5 in modulating vascular leak. Additional studies showed αvβ5 functions in the adhesion and migration of kidney pericytes in vitro. Initial studies monitoring renal blood flow after IRI did not find significant effects with αvβ5 inhibition; however, future studies should explore the contribution of vasomotor effects. These studies identify a role for αvβ5 in modulating injury-induced renal vascular leak, possibly through effects on pericyte adhesion and migration, and reveal αvβ5 inhibition as a promising therapeutic strategy for AKI.

Cells change their shape and mechanics dramatically during development and tissue healing in response to morphogens, cell-cell contact, adhesion to extracellular matrix, and more. Several regulatory links have been described between cell shape, cytoskeletal tension, matrix adhesiveness and stiffness, and recent studies have begun to uncover how these mechanotransduction pathways can impact transcriptional signaling and cell fate decision. The integrated mechanisms linking cell forces, form and fate are likely critical for driving normal morphogenesis, tissue development, and healing. Dysregulation of these mechanisms may also tip the scale from normal to diseased states. Here, we highlight mechanisms that alter cell shape and mechanics, and the pathways affected by these changes.

Contractile forces exerted on the surrounding extracellular matrix (ECM) lead to the alignment and stretching of constituent fibers within the vicinity of cells. As a consequence, the matrix reorganizes to form thick bundles of aligned fibers that enable force transmission over distances larger than the size of the cells. Contractile force-mediated remodeling of ECM fibers has bearing on a number of physiologic and pathophysiologic phenomena. In this work, we present a computational model to capture cell-mediated remodeling within fibrous matrices using finite element based discrete fiber network simulations. The model is shown to accurately capture collagen alignment, heterogeneous deformations, and long-range force transmission observed experimentally. The zone of mechanical influence surrounding a single contractile cell and the interaction between two cells are predicted from the strain-induced alignment of fibers. Through parametric studies, the effect of cell contractility and cell shape anisotropy on matrix remodeling and force transmission are quantified and summarized in a phase diagram. For highly contractile and elongated cells, we find a sensing distance that is ten times the cell size, in agreement with experimental observations., Accepted for publication in the Biophysical Journal

We developed molecular tension probes (TPs) that report traction forces of adherent cells with high spatial resolution, can in principle be linked to virtually any surface, and obviate monitoring deformations of elastic substrates. TPs consist of DNA hairpins conjugated to fluorophore-quencher pairs that unfold and fluoresce when subjected to specific forces. We applied TPs to reveal that cellular traction forces are heterogeneous within focal adhesions and localized at their distal edges.

The density and architecture of capillary beds that form within a tissue depend on many factors, including local metabolic demand and blood flow. Here, using microfluidic control of local fluid mechanics, we show the existence of a previously unappreciated flow-induced shear stress threshold that triggers angiogenic sprouting. Both intraluminal shear stress over the endothelium and transmural flow through the endothelium above 10 dyn/cm(2) triggered endothelial cells to sprout and invade into the underlying matrix, and this threshold is not impacted by the maturation of cell-cell junctions or pressure gradient across the monolayer. Antagonizing VE-cadherin widened cell-cell junctions and reduced the applied shear stress for a given transmural flow rate, but did not affect the shear threshold for sprouting. Furthermore, both transmural and luminal flow induced expression of matrix metalloproteinase 1, and this up-regulation was required for the flow-induced sprouting. Once sprouting was initiated, continuous flow was needed to both sustain sprouting and prevent retraction. To explore the potential ramifications of a shear threshold on the spatial patterning of new sprouts, we used finite-element modeling to predict fluid shear in a variety of geometric settings and then experimentally demonstrated that transmural flow guided preferential sprouting toward paths of draining interstitial fluid flow as might occur to connect capillary beds to venules or lymphatics. In addition, we show that luminal shear increases in local narrowings of vessels to trigger sprouting, perhaps ultimately to normalize shear stress across the vasculature. Together, these studies highlight the role of shear stress in controlling angiogenic sprouting and offer a potential homeostatic mechanism for regulating vascular density.

The electrophysiological consequences of cardiomyocyte and myofibroblast interactions remain unclear, and the contribution of mechanical coupling between these two cell types is still poorly understood. In this study, we examined the time course and mechanisms by which addition of myofibroblasts activated by transforming growth factor-beta (TGF-β) influence the conduction velocity (CV) of neonatal rat ventricular cell monolayers. We observed that myofibroblasts affected CV within 30 min of contact and that these effects were temporally correlated with membrane deformation of cardiomyocytes by the myofibroblasts. Expression of dominant negative RhoA in the myofibroblasts impaired both myofibroblast contraction and myofibroblast-induced slowing of cardiac conduction, whereas overexpression of constitutive RhoA had little effect. To determine the importance of mechanical coupling between these cell types, we examined the expression of the two primary cadherins in the heart (N- and OB-cadherin) at cell–cell contacts formed between myofibroblasts and cardiomyocytes. Although OB-cadherin was frequently found at myofibroblast–myofibroblast contacts, very little expression was observed at myofibroblast–cardiomyocyte contacts. The myofibroblast-induced slowing of cardiac conduction was not prevented by silencing of OB-cadherin in the myofibroblasts, and could be reversed by inhibitors of mechanosensitive channels (gadolinium or streptomycin) and cellular contraction (blebbistatin). In contrast, N-cadherin expression was commonly observed at myofibroblast–cardiomyocyte contacts, and silencing of N-cadherin in myofibroblasts prevented the myofibroblast-dependent slowing of cardiac conduction. We propose that myofibroblasts can impair the electrophysiological function of cardiac tissue through the application of contractile force to the cardiomyocyte membrane via N-cadherin junctions.

The orientation of cells and associated F-actin stress fibers is essential for proper tissue functioning. We have previously developed a computational model that qualitatively describes stress fiber orientation in response to a range of mechanical stimuli. In this paper, the aim is to quantitatively validate the model in a static, heterogeneous environment. The stress fiber orientation in uniaxially and biaxially constrained microscale tissues was investigated using a recently developed experimental system. Computed and experimental stress fiber orientations were compared, while accounting for changes in orientation with location in the tissue. This allowed for validation of the model, and additionally, it showed how sensitive the stress fiber orientation in the experimental system is to the location where it is measured, i.e., the heterogeneity of the stress fiber orientation. Computed and experimental stress fiber orientations showed good quantitative agreement in most regions. A strong local alignment near the locations where boundary conditions were enforced was observed for both uniaxially and biaxially constrained tissues. Excepting these regions, in biaxially constrained tissues, no preferred orientation was found and the distribution was independent of location. The stress fiber orientation in uniaxially constrained tissues was more heterogeneous, and stress fibers mainly oriented in the constrained direction or along the free edge. These results indicate that the stress fiber orientation in these constrained microtissues is mainly determined by the local mechanical environment, as hypothesized in our model, and also that the model is a valid tool to predict stress fiber orientation in heterogeneously loaded tissues. © 2014 Springer-Verlag Berlin Heidelberg.

Tumor dormancy is a clinical phenomenon in which disseminated tumor cells remain asymptomatic and undetectable over a long period of time. Dormant cells are able to maintain quiescence in hostile microenvironments, escape frontline cancer therapies and evade the immune system, as well as their propensity to reactivate from latency and cause metastatic relapse. Although tumor dormancy is an important problem in the treatment of cancer, the molecular mechanisms underlying this complex process remain unclear. Because dormant cells are frequently found surrounded by a laminin-rich ECM, we hypothesized that ECM dimensionality intrinsically affects cell behaviors that predispose to dormancy. Accordingly, we assessed the biophysical and biochemical response of mammary epithelial cells (MECs) to compliant polyacrylamide (PA) gels and micropatterned surfaces in which ligand presentation and ECM dimensionality were modulated to recapitulate different ECM landscapes. Using traction force microscopy, atomic force microscopy indentation, and laser ablation studies, we found that a 3D ECM led to a drop in cortical tension of MECs. Computational modeling predicted that reduced cortical tension should lead to an increase in the number and/or residence time of actin protrusions as well as a net increase in negative membrane curvature. To test the prediction, we ectopically expressed F-actin and plasma membrane markers in MECs to examine the plasma membrane topography and actin protrusion dynamics. Indeed, MECs in 3D ECM had longer and more stable actin protrusions and more negative membrane curvature inducing proteins, including Exo70, at the plasma membrane. The enrichment of Exo70 at the plasma membrane accompanied the activation of Arf6, which led to an increase in Rac/p38 pro-survival signaling pathway. Consistently, we found that the non-spread cells were able to survive in 3D ECM but the non-spread MECs plated on either a soft 2D PA gels or rigid micropatterned adhesive islands died. We next examined if reduced cortical tension is sufficient to activate pro-survival pathways in cells in 2D. Importantly, pharmacological inhibition of myosin in non-spread MECs on a 2D ECM, which causes loss of cortical tension, increased negative membrane curvature and cell viability. Conversely, genetic knockdown of the negative curvature-inducing protein Exo70 compromised MEC survival in a 3D ECM. Our results provide the first evidence demonstrating that ECM dimensionality alters the biophysical properties of cells to modulate plasma membrane curvature and activate pro-survival signaling pathways. Our findings also offer a unique perspective for why Arf6 and Rac GTPases have been implicated in cancer aggression and suggest that targeting the tissue ECM or cellular cortical tension may provide a novel therapeutic approach to target dormant cells. Citation Format: FuiBoon Kai, Guanqing Ou, Alexandra Long, Wei Guo, Richard Tourdot, Ravi Radhakrishnan, Christopher Chen, Sophie Dumont, Valerie M. Weaver. Extracellular matrix dimensionality reduces cellular cortical tension to stimulate pro-survival signaling in mammary epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1028.

Cell migration, critical to numerous biological processes, can be guided by surface topography. Studying the effects of topography on cell migration is valuable for enhancing our understanding of directional cell migration and for functionally engineering cell behavior. However, fabrication limitations constrain topography studies to geometries that may not adequately mimic physiological environments. Direct Laser Writing (DLW) provides the necessary 3D flexibility and control to create well-defined waveforms with curvature and length scales that are similar to those found in physiological settings, such as the luminal walls of blood vessels that endothelial cells migrate along. We find that endothelial cells migrate fastest along square waves, intermediate along triangular waves, and slowest along sine waves and that directional cell migration on sine waves decreases as sinusoid wavelength increases. Interestingly, inhibition of Rac1 decreases directional migration on sine wave topographies but not on flat surfaces with micropatterned lines, suggesting that cells may utilize different molecular pathways to sense curved topographies. Our study demonstrates that DLW can be employed to investigate the effects and mechanisms of topography on cell migration by fabricating a wide array of physiologically-relevant surfaces with curvatures that are challenging to fabricate using conventional manufacturing techniques.

125 Background: GLS-010 is a novel fully human anti-PD-1 mAb. Phase Ia exhibited good result of tolerance and 240mg fixed dose was selected. Phase Ib is to explore efficacy and biomarkers in different types of advanced cancer. Here we report the preliminary result in Chinese gastrointestinal (GI) tumor patients (pts) from the phase Ib. Methods: All pts enrolled received GLS-010 240mg every 2 weeks. Tumor response was assessed by RECIST 1.1 every 8 weeks. Adverse events (AEs) were graded by NCI CTCAE v4.03. Several biomarkers were evaluated, including PD-L1 by IHC, tissue tumor mutation burden (tTMB) by whole exome sequencing (WES) from FFPE tissue, blood TMB (bTMB) by the multi-gene panel based next-generation sequencing (NGS) from blood ctDNA. Results: Until September 2018, 23 pts (including 10 gastric cancer or GC, 10 esophagus cancer or EC, and 3 biliary tract cancer or BTC) were enrolled in the phase 1b. The median dosing number was 4 (range: 1~16). The most common treatment related AEs included haemoglobin decreased (16/23, G1-2), leukopenia (6/23, G1-2), fever (4/23, G1), blood bilirubin increased (4/23, G1-3), ALT increased (3/23, G1), etc. Treatment–related grade 3-5 AEs include 1 multiple organ dysfunction syndrome, 1 interstitial lung disease and 1 blood bilirubin increased. 21 pts received response evaluation. Four patients achieved partial response (PR), including 1 GC (1/9), 2 EC (2/10), and 1 BTC (1/2). 2 subjects had stable disease (SD) at Week 8, and were still in treatment. No apparent correlation was observed between treatment response and PD-L1 expression. However, both tTMB and bTMB data obtained from 18 patients support positive correlation to tumor response. The tTMB level of 3 PR pts with valid data is significantly higher than that of 9 PD pts (P = 0.036). The bTMB level of 4 PR pts with valid data is significantly higher than that of 10 PD pts (P = 0.049). Conclusions: GLS-010 showed promising efficacy and acceptable safety in Chinese patients. For Chinese GI tumor TMB may be a useful biomarker to predict the treatment response to PD-1 inhibitor. Comparing to tTMB, bTMB may be of more future value due to its applicability in clinical practice.

Rigidity sensing plays a fundamental role in multiple cell functions ranging from migration, to proliferation and differentiation (Engler et al., Cell 126:677–689, 2006; Lo et al., Biophys. J. 79:144–152, 2000; Wells, Hepatology 47:1394–1400, 2008; Zoldan et al., Biomaterials 32:9612–9621, 2011). During migration, single cells have been reported to preferentially move toward more rigid regions of a substrate in a process termed durotaxis. Durotaxis could contribute to cell migration in wound healing and gastrulation, where local gradients in tissue rigidity have been described. Despite the potential importance of this phenomenon to physiology and disease, it remains unclear how rigidity guides these behaviors and the underlying cellular and molecular mechanisms. To investigate the functional role of subcellular distribution and dynamics of cellular traction forces during durotaxis, we developed a unique microfabrication strategy to generate elastomeric micropost arrays patterned with regions exhibiting two different rigidities juxtaposed next to each other. After initial cell attachment on the rigidity boundary of the micropost array, NIH 3T3 fibroblasts were observed to preferentially migrate toward the rigid region of the micropost array, indicative of durotaxis. Additionally, cells bridging two rigidities across the rigidity boundary on the micropost array developed stronger traction forces on the more rigid side of the substrate indistinguishable from forces generated by cells exclusively seeded on rigid regions of the micropost array. Together, our results highlighted the utility of step-rigidity micropost arrays to investigate the functional role of traction forces in rigidity sensing and durotaxis, suggesting that cells could sense substrate rigidity locally to induce an asymmetrical intracellular traction force distribution to contribute to durotaxis.

The mechanical properties of the extracellular matrix (ECM) in which cells reside have emerged as an important regulator of cell fate. While materials based on natural ECM have been used to implicate the role of substrate stiffness for cell fate decisions, it is difficult in these matrices to isolate mechanics from other structural parameters. In contrast, fully synthetic hydrogels offer independent control over physical and adhesive properties. New synthetic materials that also recreate the fibrous structural hierarchy of natural matrices are now being designed to study substrate mechanics in more complex ECMs. This perspective examines the ways in which new materials are being used to advance our understanding of how extracellular matrix stiffness impacts cell function.

Electrospinning has recently gained much interest due to its ability to form scaffolds that mimic the nanofibrous nature of the extracellular matrix, such as the size and depth-dependent alignment of collagen fibers within hyaline cartilage. While much progress has been made in developing bulk, isotropic hydrogels for tissue engineering and understanding how the microenvironment of such scaffolds affects cell response, these effects have not been extensively studied in a nanofibrous system. Here, we show that the mechanics (through intrafiber crosslink density) and adhesivity (through RGD density) of electrospun hyaluronic acid (HA) fibers significantly affect human mesenchymal stem cell (hMSC) interactions and gene expression. Specifically, hMSC spreading, proliferation, and focal adhesion formation were dependent on RGD density, but not on the range of fiber mechanics investigated. Moreover, traction-mediated fiber displacements generally increased with more adhesive fibers. The expression of chondrogenic markers, unlike trends in cell spreading and cytoskeletal organization, was influenced by both fiber mechanics and adhesivity, in which softer fibers and lower RGD densities generally enhanced chondrogenesis. This work not only reveals concurrent effects of mechanics and adhesivity in a fibrous context, but also highlights fibrous HA hydrogels as a promising scaffold for future cartilage repair strategies.

Fluid shear stress (FSS) from blood flow acting on the endothelium critically regulates vascular morphogenesis, blood pressure, and atherosclerosis [1]. FSS applied to endothelial cells (ECs) triggers signaling events including opening of ion channels, activation of signaling pathways, and changes in gene expression. Elucidating how ECs sense flow is important for understanding both normal vascular function and disease. EC responses to FSS are mediated in part by a junctional mechanosensory complex consisting of VE-cadherin, PECAM-1, and VEGFR2 [2]. Previous work suggested that flow increases force on PECAM-1, which initiates signaling [2-4]. Deletion of PECAM-1 blocks responses to flow in vitro and flow-dependent vascular remodeling in vivo [2, 5]. To understand this process, we developed and validated FRET-based tension sensors for VE-cadherin and PECAM-1 using our previously developed FRET tension biosensor [6]. FRET measurements showed that in static culture, VE-cadherin in cell-cell junctions bears significant myosin-dependent tension, whereas there was no detectable tension on VE-cadherin outside of junctions. Onset of shear stress triggered a rapid (

Although calcium phosphate-containing biomaterials are promising scaffolds for bone regenerative strategies, the osteoinductive capacity of such materials is poorly understood. In this study, we investigated whether endogenous mechanisms of in vivo calcium phosphate-driven, ectopic bone formation could be identified and used to induce enhanced differentiation in vitro of the same progenitor population. To accomplish this, human periosteum derived cells (hPDCs) were seeded on hydroxyapatite/collagen scaffolds (calcium phosphate rich matrix or CPRM), or on decalcified scaffolds (calcium phosphate depleted matrix or CPDM), followed by subcutaneous implantation in nude mice to trigger ectopic bone formation. In this system, osteoblast differentiation occurred in CPRM scaffolds, but not in CPDM scaffolds. Gene expression was assessed by human full-genome microarray at 20 h after seeding, and 2, 8 and 18 days after implantation. In both matrices, implantation of the cell constructs triggered a similar gene expression cascade, however, gene expression dynamics progressed faster in CPRM scaffolds than in CPDM scaffolds. The difference in gene expression dynamics was associated with differential activation of hub genes and molecular signaling pathways related to calcium signaling (CREB), inflammation (TNFα, NFkB, and IL6) and bone development (TGFβ, β-catenin, BMP, EGF, and ERK signaling). Starting from this set of pathways, a growth factor cocktail was developed that robustly enhanced osteogenesis in vitro and in vivo. Taken together, our data demonstrate that through the identification and subsequent stimulation of genes, proteins and signaling pathways associated with calcium phosphate mediated osteoinduction, a focused approach to develop targeted differentiation protocols in adult progenitor cells can be achieved.

Stem cells have been shown to have the potential to provide a source of cells for applications to tissue engineering and organ repair. The mechanisms that regulate stem cell fate, however, mostly remain unclear. Mesenchymal stem cells (MSCs) are multipotent progenitor cells that are isolated from bone marrow and other adult tissues, and can be differentiated into multiple cell lineages, such as bone, cartilage, fat, muscles and neurons. Although previous studies have focused intensively on the effects of chemical signals that regulate MSC commitment, the effects of physical/mechanical cues of the microenvironment on MSC fate determination have long been neglected. However, several studies provided evidence that mechanical signals, both direct and indirect, played important roles in regulating a stem cell fate. In this review, we summarize a number of recent studies on how cell adhesion and mechanical cues influence the differentiation of MSCs into specific lineages. Understanding how chemical and mechanical cues in the microenvironment orchestrate stem cell differentiation may provide new insights into ways to improve our techniques in cell therapy and organ repair.

Tissue vascularization and integration with host circulation remains a key barrier to the translation of engineered tissues into clinically relevant therapies. Here, we used a microtissue molding approach to demonstrate that constructs containing highly aligned “cords” of endothelial cells triggered the formation of new capillaries along the length of the patterned cords. These vessels became perfused with host blood as early as 3 d post implantation and became progressively more mature through 28 d. Immunohistochemical analysis showed that the neovessels were composed of human and mouse endothelial cells and exhibited a mature phenotype, as indicated by the presence of alpha-smooth muscle actin–positive pericytes. Implantation of cords with a prescribed geometry demonstrated that they provided a template that defined the neovascular architecture in vivo. To explore the utility of this geometric control, we implanted primary rat and human hepatocyte constructs containing randomly organized endothelial networks vs. ordered cords. We found substantially enhanced hepatic survival and function in the constructs containing ordered cords following transplantation in mice. These findings demonstrate the importance of multicellular architecture in tissue integration and function, and our approach provides a unique strategy to engineer vascular architecture.

Angiogenesis is a complex morphogenetic process whereby endothelial cells from existing vessels invade as multicellular sprouts to form new vessels. Here, we have engineered a unique organotypic model of angiogenic sprouting and neovessel formation that originates from preformed artificial vessels fully encapsulated within a 3D extracellular matrix. Using this model, we screened the effects of angiogenic factors and identified two distinct cocktails that promoted robust multicellular endothelial sprouting. The angiogenic sprouts in our system exhibited hallmark structural features of in vivo angiogenesis, including directed invasion of leading cells that developed filopodia-like protrusions characteristic of tip cells, following stalk cells exhibiting apical–basal polarity, and lumens and branches connecting back to the parent vessels. Ultimately, sprouts bridged between preformed channels and formed perfusable neovessels. Using this model, we investigated the effects of angiogenic inhibitors on sprouting morphogenesis. Interestingly, the ability of VEGF receptor 2 inhibition to antagonize filopodia formation in tip cells was context-dependent, suggesting a mechanism by which vessels might be able to toggle between VEGF-dependent and VEGF-independent modes of angiogenesis. Like VEGF, sphingosine-1-phosphate also seemed to exert its proangiogenic effects by stimulating directional filopodial extension, whereas matrix metalloproteinase inhibitors prevented sprout extension but had no impact on filopodial formation. Together, these results demonstrate an in vitro 3D biomimetic model that reconstitutes the morphogenetic steps of angiogenic sprouting and highlight the potential utility of the model to elucidate the molecular mechanisms that coordinate the complex series of events involved in neovascularization.

Although cell-matrix adhesive interactions are known to regulate stem cell differentiation, the underlying mechanisms, in particular for direct three-dimensional (3D) encapsulation within hydrogels, are poorly understood. Here, we demonstrate that in covalently crosslinked hyaluronic acid (HA) hydrogels, the differentiation of human mesenchymal stem cells (hMSCs) is directed by the generation of degradation-mediated cellular-traction, independent of cell morphology or matrix mechanics. hMSCs within HA hydrogels of equivalent elastic moduli that either permit (restrict) cell-mediated degradation exhibited high (low) degrees of cell spreading and high (low) tractions, and favoured osteogenesis (adipogenesis). In addition, switching the permissive hydrogel to a restrictive state via delayed secondary crosslinking reduced further hydrogel degradation, suppressed traction, and caused a switch from osteogenesis to adipogenesis in the absence of changes to the extended cellular morphology. Also, inhibiting tension-mediated signalling in the permissive environment mirrored the effects of delayed secondary crosslinking, whereas upregulating tension induced osteogenesis even in the restrictive environment.

Cells dynamically assemble and organize into complex tissues during development, and the resulting three-dimensional (3D) arrangement of cells and their surrounding extracellular matrix in turn feeds back to regulate cell and tissue function. Recent advances in engineered cultures of cells to model 3D tissues or organoids have begun to capture this dynamic reciprocity between form and function. Here, we describe the underlying principles that have advanced the field, focusing in particular on recent progress in using mechanical constraints to recapitulate the structure and function of musculoskeletal tissues.

Human embryonic stem cells (hESCs) is a potential unlimited ex vivo source of ventricular (V) cardiomyocytes (CMs), but hESC-VCMs and their engineered tissues display immature traits. In adult VCMs, sarcolemmal (sarc) and mitochondrial (mito) ATP-sensitive potassium (KATP) channels play crucial roles in excitability and cardioprotection. In this study, we aim to investigate the biological roles and use of sarcKATP and mitoKATP in hESC-VCM. We showed that SarcIK, ATP in single hESC-VCMs was dormant under baseline conditions, but became markedly activated by cyanide (CN) or the known opener P1075 with a current density that was ~8-fold smaller than adult; These effects were reversible upon washout or the addition of GLI or HMR1098. Interestingly, sarcIK, ATP displayed a ~3-fold increase after treatment with hypoxia (5% O2). MitoIK, ATP was absent in hESC-VCMs. However, the thyroid hormone T3 up-regulated mitoIK, ATP, conferring diazoxide protective effect on T3-treated hESC-VCMs. When assessed using a multi-cellular engineered 3D ventricular cardiac micro-tissue (hvCMT) system, T3 substantially enhanced the developed tension by 3-folds. Diazoxide also attenuated the decrease in contractility induced by simulated ischemia (1% O2). We conclude that hypoxia and T3 enhance the functionality of hESC-VCMs and their engineered tissues by selectively acting on sarc and mitoIK, ATP.

The biomechanical behavior of tissues under mechanical stimulation is critically important to physiological function. We report a combined experimental and modeling study of bioengineered 3D smooth muscle microtissues that reveals a previously unappreciated interaction between active cell mechanics and the viscoplastic properties of the extracellular matrix. The microtissues’ response to stretch/unstretch actuations, as probed by microcantilever force sensors, was dominated by cellular actomyosin dynamics. However, cell lysis revealed a viscoplastic response of the underlying model collagen/fibrin matrix. A model coupling Hill-type actomyosin dynamics with a plastic perfectly viscoplastic description of the matrix quantitatively accounts for the microtissue dynamics, including notably the cells’ shielding of the matrix plasticity. Stretch measurements of single cells confirmed the active cell dynamics and were well described by a single-cell version of our model. These results reveal the need for new focus on matrix plasticity and its interactions with active cell mechanics in describing tissue dynamics.

Cancer metastasis is a multi-step, secondary tumor formation that is responsible for the vast majority of deaths in cancer patients. Animal models have served as one of the major tools for studying metastatic diseases. However, these metastasis models inherently lack the ability to decouple many of the key parameters that might contribute to cancer progression, and therefore ultimately limit detailed, mechanistic investigation of metastasis. Recently, organ-on-a-chip model systems have been developed for various tissue types with the potential to recapitulate major components of metastasis. Here, we discuss recent advances in in vitro biomimetic on-a-chip models for cancer metastasis.

Summary Objective This study aims to demonstrate that cartilage nanoindentation modulus is a highly sensitive indicator of the onset and spatiotemporal progression of post-traumatic osteoarthritis (PTOA) in murine models. Design Destabilization of the medial meniscus (DMM) surgery was performed on the right knees of 12-week old male, wild-type C57BL/6 mice, with Sham control on contralateral left knees. Atomic force microscopy (AFM)-based nanoindentation was applied to quantify the nanoindentation modulus, E ind , of femoral condyle cartilage at 3 days to 12 weeks after surgery. The modulus changes were compared against the timeline of histological OA signs. Meanwhile, at 8 weeks after surgery, changes in meniscus, synovium and subchondral bone were evaluated to reveal the spatial progression of PTOA. Results The modulus of medial condyle cartilage was significantly reduced at 1 week after DMM, preceding the histological OA signs, which only became detectable at 4–8 weeks after. This reduction is likely due to concomitantly elevated proteolytic activities, as blocking enzymatic activities in mice can attenuate this modulus reduction. In later OA, lateral condyle cartilage and medial meniscus also started to be weakened, illustrating the whole-organ nature of PTOA. Conclusions This study underscores the high sensitivity of nanoindentation in examining the initiation, attenuation and progression of PTOA in murine models. Meanwhile, modulus changes highlight concomitant changes in lateral cartilage and meniscus during the advancement of OA.

Mesenchymal stem cell (MSC) differentiation is mediated by soluble and physical cues. In this study, we investigated differentiation-induced transformations in MSC cellular and nuclear biophysical properties and queried their role in mechanosensation. Our data show that nuclei in differentiated bovine and human MSCs stiffen and become resistant to deformation. This attenuated nuclear deformation was governed by restructuring of Lamin A/C and increased heterochromatin content. This change in nuclear stiffness sensitized MSCs to mechanical-loading-induced calcium signaling and differentiated marker expression. This sensitization was reversed when the ‘stiff’ differentiated nucleus was softened and was enhanced when the ‘soft’ undifferentiated nucleus was stiffened through pharmacologic treatment. Interestingly, dynamic loading of undifferentiated MSCs, in the absence of soluble differentiation factors, stiffened and condensed the nucleus, and increased mechanosensitivity more rapidly than soluble factors. These data suggest that the nucleus acts as a mechanostat to modulate cellular mechanosensation during differentiation. DOI: http://dx.doi.org/10.7554/eLife.18207.001

Objective: Angiogenesis is crucial to maintain tissue homeostasis under nutrient and oxygen deprivation (ischemia). Although considerable evidence supports that angiogenesis is regulated by hypoxia-HIF1α induction of vascular endothelial growth factor (VEGF), the role of nutrient deprivation in angiogenesis is poorly defined. Approach and Results: We report that nutrient deprivation in the form of dietary sulfur amino acid restriction (Methionine/cysteine Restriction; MR) promotes VEGF expression and functional growth of new capillaries in skeletal muscle of mice (Fig.1 A, B). This occurred independently of hypoxia or HIF1α, but instead required the amino acid-sensing eIF2α kinase GCN2 and the transcription factor ATF4 (Fig. 1C). In addition to increased VEGF, nutrient deprivation boosted production of the pro-angiogenic gas hydrogen sulfide (H 2 S) via increased GCN2/ATF4-dependent expression of the H 2 S-generating enzyme cystathionine-gamma-lyase (CGL). The genetic requirement for CGL in angiogenesis triggered by nutrient deprivation, exercise or local VEGF overexpression, as well as the ability of local CGL overexpression to promote angiogenesis in vivo, revealed the critical importance of CGL-derived H 2 S in angiogenesis (Fig. 1D). Finally, plasma H 2 S was reduced in patients with vascular disease (versus non-diseased age-matched controls), and correlated with 2-year survival following vascular surgery (Fig.1 E, F). Conclusions: These data reveal a nutrient-sensing pathway targetable by diet as a previously unrecognized central regulator of VEGF expression and angiogenesis independent of canonical hypoxic signaling. This discovery points to novel dietary interventions and GCN2/ATF4/CGL/H 2 S-based strategies to manipulate angiogenesis. Figure 1

The effects of hemodynamic and interstitial mechanical forces on endothelial biology in vivo have been appreciated for over half a century, regulating vessel network development, homeostatic function, and progression of vascular disease. Investigations using cultures of endothelial cells on two-dimensional (2D) substrates have elucidated important mechanisms by which microenvironmental stresses are sensed and transduced into chemical signaling responses. However recent studies in vivo and in three-dimensional (3D) in vitro models of vascular beds have enabled the investigation of forces and cellular behaviors previously not possible in traditional 2D culture systems. These studies support a developing paradigm that the 3D chemo-mechanical architecture of the vascular niche impacts how endothelial cells both sense and respond to microenvironmental forces. We present evolving concepts in endothelial force sensing and mechanical signaling and highlight recent insights gained from in vivo and 3D in vitro vascular models.

The major function of the lymphatic system is to drain interstitial fluid from tissue. Functional drainage causes increased fluid flow that triggers lymphatic expansion, which is conceptually similar to hypoxia-triggered angiogenesis. Here, we have identified a mechanotransduction pathway that translates laminar flow-induced shear stress to activation of lymphatic sprouting. While low-rate laminar flow commonly induces the classic shear stress responses in blood endothelial cells and lymphatic endothelial cells (LECs), only LECs display reduced Notch activity and increased sprouting capacity. In response to flow, the plasma membrane calcium channel ORAI1 mediates calcium influx in LECs and activates calmodulin to facilitate a physical interaction between Krüppel-like factor 2 (KLF2), the major regulator of shear responses, and PROX1, the master regulator of lymphatic development. The PROX1/KLF2 complex upregulates the expression of DTX1 and DTX3L. DTX1 and DTX3L, functioning as a heterodimeric Notch E3 ligase, concertedly downregulate NOTCH1 activity and enhance lymphatic sprouting. Notably, overexpression of the calcium reporter GCaMP3 unexpectedly inhibited lymphatic sprouting, presumably by disturbing calcium signaling. Endothelial-specific knockouts of Orai1 and Klf2 also markedly impaired lymphatic sprouting. Moreover, Dtx3l loss of function led to defective lymphatic sprouting, while Dtx3l gain of function rescued impaired sprouting in Orai1 KO embryos. Together, the data reveal a molecular mechanism underlying laminar flow-induced lymphatic sprouting.

Cadherins and integrins are intrinsically linked through the actin cytoskeleton and share common signaling molecules. Although mechanosensing by the integrin–actin axis has long been appreciated, a growing body of literature now demonstrates that cadherins also transduce and respond to mechanical forces. Mounting evidence shows that mechanically driven crosstalk between integrins and cadherins regulates the spatial distribution of these receptors, their signaling intermediates, the actin cytoskeleton and intracellular forces. This interplay between integrins and cadherins can control fibronectin matrix assembly and signaling, and a fine balance between traction forces at focal adhesions and intercellular tension at adherens junctions is crucial for directional collective cell migration. In this Commentary, we discuss two central ideas: (1) how the dynamic interplay between integrins and cadherins regulates the spatial organization of intracellular signals and the extracellular matrix, and (2) the emerging consensus that intracellular force is a central mechanism that dictates cell behavior, guides tissue development and ultimately drives physiology.

Recent methods have revealed that cells on planar substrates exert both shear (in-plane) and normal (out-of-plane) tractions against the extracellular matrix (ECM). However, the location and origin of the normal tractions with respect to the adhesive and cytoskeletal elements of cells have not been elucidated. We developed a high-spatiotemporal-resolution, multidimensional (2.5D) traction force microscopy to measure and model the full 3D nature of cellular forces on planar 2D surfaces. We show that shear tractions are centered under elongated focal adhesions whereas upward and downward normal tractions are detected on distal (toward the cell edge) and proximal (toward the cell body) ends of adhesions, respectively. Together, these forces produce significant rotational moments about focal adhesions in both protruding and retracting peripheral regions. Temporal 2.5D traction force microscopy analysis of migrating and spreading cells shows that these rotational moments are highly dynamic, propagating outward with the leading edge of the cell. Finally, we developed a finite element model to examine how rotational moments could be generated about focal adhesions in a thin lamella. Our model suggests that rotational moments can be generated largely via shear lag transfer to the underlying ECM from actomyosin contractility applied at the intracellular surface of a rigid adhesion of finite thickness. Together, these data demonstrate and probe the origin of a previously unappreciated multidimensional stress profile associated with adhesions and highlight the importance of new approaches to characterize cellular forces.

Summary Background The ternary complex factors (TCFs; Elk1, Net, and Sap-1) are growth factor-responsive transcription cofactors of serum response factor (SRF) and are activated by MAP kinase (MAPK) phosphorylation to regulate immediate early gene transcription. Although cell adhesion also can regulate immediate early genes and proliferation, the mechanism for this effect has remained unexplored. Results Restricting adhesion and spreading of G 0 -synchronized cells on substrates with decreasing size of micropatterned islands of fibronectin suppressed serum-induced immediate early gene expression and S phase entry. Knockdown of Sap-1 decreased expression of the immediate early genes egr1 and fos and subsequent proliferation normally present with high adhesion, whereas knockdown of Net rescued egr1 and fos expression and proliferation normally suppressed by low adhesion. Chromatin immunoprecipitation studies showed increased occupancy of egr1 and fos promoters by Sap-1 with high adhesion, whereas low adhesion increased Net occupancy. This switch in TCF promoter binding was regulated by an adhesion-mediated switch in MAPK activity. Increasing adhesion enhanced serum-induced JNK activity while suppressing p38 activity, leading to increased Sap-1 phosphorylation and Net dephosphorylation, and switching Net with Sap-1 at egr1 and fos promoters to support proliferation. Microarray studies confirmed this switch in TCF regulation of proliferative genes and uncovered novel gene targets and functions coregulated by Sap-1 and Net. Conclusions These data demonstrate a key role for the TCFs in adhesion-induced transcription and proliferation and reveal a novel MAPK/TCF transcriptional switch that controls this process.

It has previously been demonstrated that cell shape can influence commitment of human bone marrow-derived mesenchymal stem cells (hBMCs) to adipogenic, osteogenic, chondrogenic, and other lineages. Human periosteum-derived cells (hPDCs) exhibit multipotency similar to hBMCs, but hPDCs may offer enhanced potential for osteogenesis and chondrogenesis given their apparent endogenous role in bone and cartilage repair in vivo. Here, we examined whether hPDC differentiation is regulated by adhesive and mechanical cues comparable to that reported for hBMC differentiation. When cultured in the appropriate induction media, hPDCs at high cell seeding density demonstrated enhanced levels of adipogenic or chondrogenic markers as compared with hPDCs at low cell seeding density. Cell seeding density correlated inversely with projected area of cell spreading, and directly limiting cell spreading with micropatterned substrates promoted adipogenesis or chondrogenesis while substrates promoting cell spreading supported osteogenesis. Interestingly, cell seeding density influenced differentiation through both changes in cell shape and non-shape-mediated effects: density-dependent adipogenesis and chondrogenesis were regulated primarily by cell shape whereas non-shape effects strongly influenced osteogenic potential. Inhibition of cytoskeletal contractility by adding the Rho kinase inhibitor Y27632 further enhanced adipogenic differentiation and discouraged osteogenic differentiation of hPDCs. Together, our results suggest that multipotent lineage decisions of hPDCs are impacted by cell adhesive and mechanical cues, though to different extents than hBMCs. Thus, future studies of hPDCs and other primary stem cell populations with clinical potential should consider varying biophysical metrics for more thorough optimization of stem cell differentiation. ispartof: Biology Open vol:1 issue:11 pages:1058-68 ispartof: location:England status: published

In the absence of perfusable vascular networks, three-dimensional (3D) engineered tissues densely populated with cells quickly develop a necrotic core [1]. Yet the lack of a general approach to rapidly construct such networks remains a major challenge for 3D tissue culture [2–4]. Here, we 3D printed rigid filament networks of carbohydrate glass, and used them as a cytocompatible sacrificial template in engineered tissues containing living cells to generate cylindrical networks which could be lined with endothelial cells and perfused with blood under high-pressure pulsatile flow. Because this simple vascular casting approach allows independent control of network geometry, endothelialization, and extravascular tissue, it is compatible with a wide variety of cell types, synthetic and natural extracellular matrices (ECMs), and crosslinking strategies. We also demonstrated that the perfused vascular channels sustained the metabolic function of primary rat hepatocytes in engineered tissue constructs that otherwise exhibited suppressed function in their core.

Osteogenic differentiation of human mesenchymal stem cells (hMSCs) is classically thought to be mediated by different cytokines such as the bone morphogenetic proteins (BMPs). Here, we report that cell adhesion to extracellular matrix (ECM), and its effects on cell shape and cytoskeletal mechanics, regulates BMP-induced signaling and osteogenic differentiation of hMSCs. Using micropatterned substrates to progressively restrict cell spreading and flattening against ECM, we demonstrated that BMP-induced osteogenesis is progressively antagonized with decreased cell spreading. BMP triggered rapid and sustained RhoA/Rho-associated protein kinase (ROCK) activity and contractile tension only in spread cells, and this signaling was required for BMP-induced osteogenesis. Exploring the molecular basis for this effect, we found that restricting cell spreading, reducing ROCK signaling, or inhibiting cytoskeletal tension prevented BMP-induced SMA/mothers against decapentaplegic (SMAD)1 c-terminal phosphorylation, SMAD1 dimerization with SMAD4, and SMAD1 translocation into the nucleus. Together, these findings demonstrate the direct involvement of cell spreading and RhoA/ROCK-mediated cytoskeletal tension generation in BMP-induced signaling and early stages of in vitro osteogenesis, and highlight the essential interplay between biochemical and mechanical cues in stem cell differentiation.

Matrix rigidity regulates a switch between TGF-β1–induced cell functions in two epithelial cell lines. On compliant polyacrylamide gels, TGF-β1 induced apoptosis, whereas on rigid gels, cells underwent an epithelial–mesenchymal transition (EMT). Compliant gels reduced PI3K/Akt activity, which was essential for cell survival and EMT on rigid gels., The transforming growth factor-β (TGF-β) signaling pathway is often misregulated during cancer progression. In early stages of tumorigenesis, TGF-β acts as a tumor suppressor by inhibiting proliferation and inducing apoptosis. However, as the disease progresses, TGF-β switches to promote tumorigenic cell functions, such as epithelial–mesenchymal transition (EMT) and increased cell motility. Dramatic changes in the cellular microenvironment are also correlated with tumor progression, including an increase in tissue stiffness. However, it is unknown whether these changes in tissue stiffness can regulate the effects of TGF-β. To this end, we examined normal murine mammary gland cells and Madin–Darby canine kidney epithelial cells cultured on polyacrylamide gels with varying rigidity and treated with TGF-β1. Varying matrix rigidity switched the functional response to TGF-β1. Decreasing rigidity increased TGF-β1–induced apoptosis, whereas increasing rigidity resulted in EMT. Matrix rigidity did not change Smad signaling, but instead regulated the PI3K/Akt signaling pathway. Direct genetic and pharmacologic manipulations further demonstrated a role for PI3K/Akt signaling in the apoptotic and EMT responses. These findings demonstrate that matrix rigidity regulates a previously undescribed switch in TGF-β–induced cell functions and provide insight into how changes in tissue mechanics during disease might contribute to the cellular response to TGF-β.

Dendritic cells (DCs) migrate from sites of inflammation to secondary lymphoid organs where they initiate the adaptive immune response. Although motility is essential to DC function, the mechanisms by which they migrate are not fully understood. We incorporated micropost array detectors into a microfluidic gradient generator to develop what we consider to be a novel method for probing low magnitude traction forces during directional migration. We found migration of primary murine DCs is driven by short-lived traction stresses at the leading edge or filopodia. The traction forces generated by DCs are smaller in magnitude than found in neutrophils, and of similar magnitude during chemotaxis and chemokinesis, at 18 ± 1.4 and 16 ± 1.3 nN/cell, respectively. The characteristic duration of local DC traction forces was 3 min. The maximum principal stress in the cell occurred in the plane perpendicular to the axis of motion, forward of the centroid. We illustrate that the spatiotemporal pattern of traction stresses can be used to predict the direction of future DC motion. Overall, DCs show a mode of migration distinct from both mesenchymal cells and neutrophils, characterized by rapid turnover of traction forces in leading filopodia.

The identification of factors that define adipocyte precursor potential has important implications for obesity. Preadipocytes are fibroblastoid cells committed to becoming round lipid-laden adipocytes. In vitro, this differentiation process is facilitated by confluency, followed by adipogenic stimuli. During adipogenesis, a large number of cytostructural genes are repressed before adipocyte gene induction. Here we report that the transcriptional repressor transcription factor 7-like 1 (TCF7L1) binds and directly regulates the expression of cell structure genes. Depletion of TCF7L1 inhibits differentiation, because TCF7L1 indirectly induces the adipogenic transcription factor peroxisome proliferator-activated receptor γ in a manner that can be replaced by inhibition of myosin II activity. TCF7L1 is induced by cell contact in adipogenic cell lines, and ectopic expression of TCF7L1 alleviates the confluency requirement for adipocytic differentiation of precursor cells. In contrast, TCF7L1 is not induced during confluency of non-adipogenic fibroblasts, and, remarkably, forced expression of TCF7L1 is sufficient to commit non-adipogenic fibroblasts to an adipogenic fate. These results establish TCF7L1 as a transcriptional hub coordinating cell–cell contact with the transcriptional repression required for adipogenic competency.

Angiogenesis is regulated by both soluble growth factors and cellular interactions with the extracellular matrix (ECM). While cell adhesion via integrins has been shown to be required for angiogenesis, the effects of quantitative changes in cell adhesion and spreading against the ECM remain less clear. Here, we show that angiogenic sprouting in natural and engineered three-dimensional matrices exhibited a biphasic response, with peak sprouting when adhesion to the matrix was limited to intermediate levels. Examining changes in global gene expression to determine a genetic basis for this response, we demonstrate a vascular endothelial growth factor (VEGF)-induced upregulation of genes associated with vascular invasion and remodeling when cell adhesion was limited, whereas cells on highly adhesive surfaces upregulated genes associated with proliferation. To explore a mechanistic basis for this effect, we turned to focal adhesion kinase (FAK), a central player in adhesion signaling previously implicated in angiogenesis, and its homologue, proline-rich tyrosine kinase 2 (Pyk2). While FAK signaling had some impact, our results suggested that Pyk2 can regulate both gene expression and endothelial sprouting through its enhanced activation by VEGF in limited adhesion contexts. We also demonstrate decreased sprouting of tissue explants from Pyk2-null mice as compared to wild type mice as further confirmation of the role of Pyk2 in angiogenic sprouting. These results suggest a surprising finding that limited cell adhesion can enhance endothelial responsiveness to VEGF and demonstrate a novel role for Pyk2 in the adhesive regulation of angiogenesis.

Understanding the dynamics of a hereditary symbiosis requires testing how ecological factors alter not only the fitness consequences of the symbiosis, but also the rate of symbiont transmission to the next generation. The relative importance of these two mechanisms remains unresolved because studies have not simultaneously examined how the ecological context of the symbiosis influences both costs/benefits and the rate of vertical transmission. Fungal endophytes in grasses have provided particularly tractable systems for investigating the ecological and evolutionary dynamics of hereditary symbiosis. Here we examine interactions between a fungal endophyte, Epichloe amarillans, and its grass host, Agrostis hyemalis, under altered abiotic and biotic contexts: a gradient of water availability and in the presence versus absence of soil microbes. We show that benefits of the symbiosis were strongest when water was limiting. Symbiotic plants at the lowest watering level produced ∼40% more inflorescences and greater seed mass than non-symbiotic plants, while at the highest watering level, symbiotic and non-symbiotic plants did not significantly differ in reproductive fitness. Benefits appear to accrue by allowing hosts to escape from drought, a response that has not been previously reported to be endophyte-mediated. Symbiotic plants at the lowest watering level flowered 9 days earlier than non-symbiotic plants. Interestingly, our results suggest the symbiosis may be costly in the presence of soil microbes, as on live soil, the biomass of symbiotic plants was lower than the biomass of symbiont-free plants. We detected no effect of either the biotic or abiotic context on the rate of symbiont vertical transmission, suggesting that the context-dependent benefits of the symbiosis are the more important driver of variation in symbiont frequency in this system.

Mechanical traction forces exerted by adherent cells on their surroundings serve an important role in a multitude of cellular and physiological processes including cell motility and multicellular rearrangements. For endothelial cells, contraction also provides a means to disrupt cell-cell junctions during inflammation to increase permeability between blood and interstitial tissue compartments. The degree of contractility exhibited by endothelial cells is influenced by numerous soluble factors, such as thrombin, histamine, lysophosphatidic acid, sphingosine-1-phosphate, and vascular endothelial growth factor (VEGF). Upon binding to cell surface receptors, these agents trigger changes in cytoskeletal organization, adhesion and myosin II activity to varying degrees. While conventional antibody-based biochemical assays are suitable for detecting relatively large changes in biomarkers of contractility in an end-point format, they cannot resolve subtle or rapid changes in contractility and cannot do so noninvasively. To overcome these limitations, we developed an approach to measure the contractile response of single cells exposed to contractility agonists with high spatiotemporal resolution. A previously developed traction force sensor, comprised of dense arrays of elastomeric microposts on which cells are cultured, was combined with custom, semi-automated software developed here to extract strain energy measurements from thousands of time-lapse images of micropost arrays deformed by adherent cells. Using this approach we corroborated the differential effects of known agonists of contractility and characterized the dynamics of their effects. All of these agonists produced a characteristic first-order rise and plateau in forces, except VEGF, which stimulated an early transient spike in strain energy followed by a sustained increase. This novel, two-phase contractile response was present in a subpopulation of cells, was mediated through both VEGFR2 and ROCK activation, and its magnitude was modulated by receptor internalization. Interestingly, the concentration of VEGF could shift the proportion of cells that responded with a spike versus only a gradual increase in forces. Furthermore, cells repeatedly exposed to VEGF were found to contract with different dynamics after pretreatment, suggesting that exposure history can impact the mechanical response. These studies highlight the importance of direct measurements of traction force dynamics as a tool for studies of mechanotransduction.

We present a novel approach to examine cell migration using dynamically adhesive substrates consisting of three spatially and functionally distinct regions: the first is permanently nonadhesive to cells, the second is permanently adhesive, and the final region is electrochemically switched from nonadhesive to adhesive. We applied a double microcontact printing approach to pattern gold surfaces with carboxylic acid-terminated self-assembled monolayers (SAMs) that permit initial cell adhesion, with methyl-terminated SAMs that permit adsorption of a nonadhesive, and with tri(ethylene glycol)-terminated SAMs that can be electrochemically "switched" to permit cell migration from a prespecified pattern onto a new pattern. Using these substrates, we investigated the migration of epithelial cells from monolayers onto narrow, branching tracks of extracellular matrix in order to characterize how lead cells influence the direction of movement of followers. Time-lapse imaging revealed that, on average, five cells consistently chose one branch before other cells entered the second branch, providing evidence to suggest that intercellular communication plays an important role in guiding the cohesive movement of epithelial sheets. This platform may be of use in furthering our understanding of the mechanisms underlying cellular decision-making during migration in both individual and multicellular contexts.