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In wound healing, tissue growth, and certain cancers, the epithelial or the endothelial monolayer sheet expands. Within the expanding monolayer sheet, migration of the individual cell is strongly guided by physical forces imposed by adjacent cells. This process is called plithotaxis and was discovered using Monolayer Stress Microscopy (MSM). MSM rests upon certain simplifying assumptions, however, concerning boundary conditions, cell material properties and system dimensionality. To assess the validity of these assumptions and to quantify associated errors, here we report new analytical, numerical, and experimental investigations. For several commonly used experimental monolayer systems, the simplifying assumptions used previously lead to errors that are shown to be quite small. Out-of-plane components of displacement and traction fields can be safely neglected, and characteristic features of intercellular stresses that underlie plithotaxis remain largely unaffected. Taken together, these findings validate Monolayer Stress Microscopy within broad but well-defined limits of applicability.

The processes by which an organism develops its shape and heals wounds involve expansion of a monolayer sheet of cells. The mechanism underpinning this epithelial expansion remains obscure, despite the fact that its failure is known to contribute to several diseases, including carcinomas, which account for about 90% of all human cancers. Here, using the micropatterned epithelial monolayer as a model system, we report the discovery of a mechanical wave that propagates slowly to span the monolayer, traverses intercellular junctions in a cooperative manner and builds up differentials of mechanical stress. Essential features of this wave generation and propagation are captured by a minimal model based on sequential fronts of cytoskeletal reinforcement and fluidization. These findings establish a mechanism of long-range cell guidance, symmetry breaking and pattern formation during monolayer expansion. Tissue growth and regrowth rely on the collective migration of sheets of cells. Gradients in tension established through intercellular forces guide this migration, but the mechanism driving the gradients has remained unclear. Innovative experiments now reveal their origin—in a mechanical wave set up by sequential cell reinforcement and fluidization.

Collective migration of endothelial cells is important for wound healing and angiogenesis. During such migration, each constituent endothelial cell coordinates its magnitude and direction of migration with its neighbors while retaining intercellular adhesion. Ensuring coordination and cohesion involves a variety of intra- and inter-cellular signaling processes. However, the role of permeation of extracellular Na + in collective cell migration remains unclear. Here, we examined the effect of Na + permeation in collective migration of pulmonary artery endothelial cell (PAEC) monolayers triggered by either a scratch injury or a barrier removal over 24 hours. In the scratch assay, PAEC monolayers migrated in two approximately linear phases. In the first phase, wound closure started with fast speed which then rapidly reduced within 5 hours after scratching. In the second phase, wound closure maintained at slow and stable speed from 6 to 24 hours. In the absence of extracellular Na + , the wound closure distance was reduced by >50%. Fewer cells at the leading edge protruded prominent lamellipodia. Beside transient gaps, some sustained interendothelial gaps also formed and progressively increased in size over time, and some fused with adjacent gaps. In the absence of both Na + and scratch injury, PAEC monolayer migrated even more slowly, and interendothelial gaps obviously increased in size towards the end. Pharmacological inhibition of the epithelial Na + channel (ENaC) using amiloride reduced wound closure distance by 30%. Inhibition of both the ENaC and the Na + /Ca 2+ exchanger (NCX) using benzamil further reduced wound closure distance in the second phase and caused accumulation of floating particles in the media. Surprisingly, pharmacological inhibition of the Ca 2+ release-activated Ca 2+ (CRAC) channel protein 1 (Orai1) using GSK-7975A, the transient receptor potential channel protein 1 and 4 (TRPC1/4) using Pico145, or both Orai1 and TRPC1/4 using combined GSK-7975A and Pico145 treatment did not affect wound closure distance dramatically. Nevertheless, the combined treatment appeared to cause accumulation of floating particles. Note that GSK-7975A also inhibits small inward Ca 2+ currents via Orai2 and Orai3 channels, whereas Pico145 also blocks TRPC4, TRPC5, and TRPC1/5 channels. By contrast, gene silence of Orai1 by shRNAs led to a 25% reduction of wound closure in the first 6 hours but had no effect afterwards. However, in the absence of extracellular Na + or cellular injury, Orai1 did not affect PAEC collective migration. Overall, the data reveal that Na + permeation into cells contributes to PAEC monolayer collective migration by increasing lamellipodial formation, reducing accumulation of floating particles, and improving intercellular adhesion.

As a wound heals, or a body plan forms, or a tumour invades, observed cellular motions within the advancing cell swarm are thought to stem from yet to be observed physical stresses that act in some direct and causal mechanical fashion. Here we show that such a relationship between motion and stress is far from direct. Using monolayer stress microscopy, we probed migration velocities, cellular tractions and intercellular stresses in an epithelial cell sheet advancing towards an island on which cells cannot adhere. We found that cells located near the island exert tractions that pull systematically towards this island regardless of whether the cells approach the island, migrate tangentially along its edge, or paradoxically, recede from it. This unanticipated cell-patterning motif, which we call kenotaxis, represents the robust and systematic mechanical drive of the cellular collective to fill unfilled space. Although the collective cellular motion involved in, for example, wound healing and tumour invasion is suspected to be driven by mechanical stresses within the advancing cell monolayer, how motion and stress relate has remained elusive. Now, stress-microscopy observations of an epithelial cell sheet advancing towards a region where cells cannot adhere reveal that the cells located nearby such a region exert forces that pull them towards the unfilled space, regardless of whether the cells approach or recede from it.

From coffee beans flowing in a chute to cells remodelling in a living tissue, a wide variety of close-packed collective systems—both inert and living—have the potential to jam. The collective can sometimes flow like a fluid or jam and rigidify like a solid. The unjammed-to-jammed transition remains poorly understood, however, and structural properties characterizing these phases remain unknown. Using primary human bronchial epithelial cells, we show that the jamming transition in asthma is linked to cell shape, thus establishing in that system a structural criterion for cell jamming. Surprisingly, the collapse of critical scaling predicts a counter-intuitive relationship between jamming, cell shape and cell–cell adhesive stresses that is borne out by direct experimental observations. Cell shape thus provides a rigorous structural signature for classification and investigation of bronchial epithelial layer jamming in asthma, and potentially in any process in disease or development in which epithelial dynamics play a prominent role. Cell shape provides a structural signature for the classification and investigation of the jamming of bronchial epithelial layers in asthma.

Cells comprising a tissue migrate as part of a collective. How collective processes are coordinated over large multi-cellular assemblies has remained unclear, however, because mechanical stresses exerted at cell–cell junctions have not been accessible experimentally. We report here maps of these stresses within and between cells comprising a monolayer. Within the cell sheet there arise unanticipated fluctuations of mechanical stress that are severe, emerge spontaneously, and ripple across the monolayer. Within that stress landscape, local cellular migrations follow local orientations of maximal principal stress. Migrations of both endothelial and epithelial monolayers conform to this behaviour, as do breast cancer cell lines before but not after the epithelial–mesenchymal transition. Collective migration in these diverse systems is seen to be governed by a simple but unifying physiological principle: neighbouring cells join forces to transmit appreciable normal stress across the cell–cell junction, but migrate along orientations of minimal intercellular shear stress. The mechanical stresses within and between cells inside an advancing cellular monolayer are mapped experimentally. Cellular migration is found to be oriented in the direction of maximum principal stress indicating that cells collectively migrate to maintain minimal local intercellular shear stress.

Pulmonary arterial hypertension (PAH) is characterized by endothelial cell (EC) dysfunction. There are no data from living patients to inform whether differential gene expression of pulmonary artery ECs (PAECs) can discern disease subtypes, progression and pathogenesis. We aimed to further validate our previously described method to propagate ECs from right heart catheter (RHC) balloon tips and to perform additional PAEC phenotyping. We performed bulk RNA sequencing of PAECs from RHC balloons. Using unsupervised dimensionality reduction and clustering we compared transcriptional signatures from PAH to controls and other forms of pulmonary hypertension. Select PAEC samples underwent single cell and population growth characterization and anoikis quantification. Fifty-four specimens were analyzed from 49 subjects. The transcriptome appeared stable over limited passages. Six genes involved in sex steroid signaling, metabolism, and oncogenesis were significantly upregulated in PAH subjects as compared to controls. Genes regulating BMP and Wnt signaling, oxidative stress and cellular metabolism were differentially expressed in PAH subjects. Changes in gene expression tracked with clinical events in PAH subjects with serial samples over time. Functional assays demonstrated enhanced replication competency and anoikis resistance. Our findings recapitulate fundamental biological processes of PAH and provide new evidence of a cancer-like phenotype in ECs from the central vasculature of PAH patients. This “cell biopsy” method may provide insight into patient and lung EC heterogeneity to advance precision medicine approaches in PAH.

Collective migration of endothelial cells is important for wound healing and angiogenesis. During such migration, each constituent endothelial cell coordinates its magnitude and direction of migration with its neighbors while retaining intercellular adhesion. Ensuring coordination and cohesion involves a variety of intra- and inter-cellular signaling processes. However, the role of permeation of extracellular Na.sup.+ in collective cell migration remains unclear. Here, we examined the effect of Na.sup.+ permeation in collective migration of pulmonary artery endothelial cell (PAEC) monolayers triggered by either a scratch injury or a barrier removal over 24 hours. In the scratch assay, PAEC monolayers migrated in two approximately linear phases. In the first phase, wound closure started with fast speed which then rapidly reduced within 5 hours after scratching. In the second phase, wound closure maintained at slow and stable speed from 6 to 24 hours. In the absence of extracellular Na.sup.+, the wound closure distance was reduced by >50%. Fewer cells at the leading edge protruded prominent lamellipodia. Beside transient gaps, some sustained interendothelial gaps also formed and progressively increased in size over time, and some fused with adjacent gaps. In the absence of both Na.sup.+ and scratch injury, PAEC monolayer migrated even more slowly, and interendothelial gaps obviously increased in size towards the end. Pharmacological inhibition of the epithelial Na.sup.+ channel (ENaC) using amiloride reduced wound closure distance by 30%. Inhibition of both the ENaC and the Na.sup.+ /Ca.sup.2+ exchanger (NCX) using benzamil further reduced wound closure distance in the second phase and caused accumulation of floating particles in the media. Surprisingly, pharmacological inhibition of the Ca.sup.2+ release-activated Ca.sup.2+ (CRAC) channel protein 1 (Orai1) using GSK-7975A, the transient receptor potential channel protein 1 and 4 (TRPC1/4) using Pico145, or both Orai1 and TRPC1/4 using combined GSK-7975A and Pico145 treatment did not affect wound closure distance dramatically. Nevertheless, the combined treatment appeared to cause accumulation of floating particles. Note that GSK-7975A also inhibits small inward Ca.sup.2+ currents via Orai2 and Orai3 channels, whereas Pico145 also blocks TRPC4, TRPC5, and TRPC1/5 channels. By contrast, gene silence of Orai1 by shRNAs led to a 25% reduction of wound closure in the first 6 hours but had no effect afterwards. However, in the absence of extracellular Na.sup.+ or cellular injury, Orai1 did not affect PAEC collective migration. Overall, the data reveal that Na.sup.+ permeation into cells contributes to PAEC monolayer collective migration by increasing lamellipodial formation, reducing accumulation of floating particles, and improving intercellular adhesion.

Pulmonary artery endothelial cells (PAECs) from patients with pulmonary arterial hypertension (PAH) exhibit heterogeneous responses to their mechanical microenvironment. PAH is characterized by pulmonary artery stiffening and vascular remodeling. In this study, the researchers investigated the mechanosensing properties of PAECs obtained from living patients with PAH. They hypothesized that the mechanical properties of the substrate may influence PAH EC characteristics and correlate with clinical disease severity. PAECs were cultured on substrates with varying stiffness and their morphology and migration were analyzed. The results showed that PAECs from different PAH subtypes exhibited distinct responses to substrate stiffness. The stiffness at which cells demonstrated maximum speed correlated with clinical features such as pulmonary vascular resistance and 6-minute walk distance. These findings suggest that PAEC phenotype can be assessed based on their response to the mechanical microenvironment and may provide insights into the heterogeneity of PAH. Further studies are needed to validate these observations and understand the underlying molecular mechanisms.

The sorting of distinctly different cell types into specific tissue compartments has long been thought to be a problem in minimization of total free energy in immiscible fluids, wherein cell–cell adhesion, cell stiffness, and cell contraction combine to define an effective macroscopic tissue surface tension. Pawlizak et al (2015 New J. Phys. 17 083049) now show not only that adhesion forces at interfaces unexpectedly fail to correlate with the density of adhesion molecules, but also that certain cancer cell lines unexpectedly fail to behave as a fluid, with cells becoming kinetically trapped in what might be a jammed, solid-like non-equilibrium state.