The mechanisms by which physical forces regulate endothelial cells to determine the complexities of vascular structure and function are enigmatic. Studies of sensory neurons have suggested Piezo proteins as subunits of Ca(2+)-permeable non-selective cationic channels for detection of noxious mechanical impact. Here we show Piezo1 (Fam38a) channels as sensors of frictional force (shear stress) and determinants of vascular structure in both development and adult physiology. Global or endothelial-specific disruption of mouse Piezo1 profoundly disturbed the developing vasculature and was embryonic lethal within days of the heart beating. Haploinsufficiency was not lethal but endothelial abnormality was detected in mature vessels. The importance of Piezo1 channels as sensors of blood flow was shown by Piezo1 dependence of shear-stress-evoked ionic current and calcium influx in endothelial cells and the ability of exogenous Piezo1 to confer sensitivity to shear stress on otherwise resistant cells. Downstream of this calcium influx there was protease activation and spatial reorganization of endothelial cells to the polarity of the applied force. The data suggest that Piezo1 channels function as pivotal integrators in vascular biology.

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Piezo1 integration of vascular architecture with physiological force

Epithelial tissues (epithelia) remove excess cells through extrusion, preventing the accumulation of unnecessary or pathological cells. The extrusion process can be triggered by apoptotic signalling, oncogenic transformation and overcrowding of cells. Despite the important linkage of cell extrusion to developmental, homeostatic and pathological processes such as cancer metastasis, its underlying mechanism and connections to the intrinsic mechanics of the epithelium are largely unexplored. We approach this problem by modelling the epithelium as an active nematic liquid crystal (that has a long range directional order), and comparing numerical simulations to strain rate and stress measurements within monolayers of MDCK (Madin Darby canine kidney) cells. Here we show that apoptotic cell extrusion is provoked by singularities in cell alignments in the form of comet-shaped topological defects. We find a universal correlation between extrusion sites and positions of nematic defects in the cell orientation field in different epithelium types. The results confirm the active nematic nature of epithelia, and demonstrate that defect-induced isotropic stresses are the primary precursors of mechanotransductive responses in cells, including YAP (Yes-associated protein) transcription factor activity, caspase-3-mediated cell death, and extrusions. Importantly, the defect-driven extrusion mechanism depends on intercellular junctions, because the weakening of cell-cell interactions in an α-catenin knockdown monolayer reduces the defect size and increases both the number of defects and extrusion rates, as is also predicted by our model. We further demonstrate the ability to control extrusion hotspots by geometrically inducing defects through microcontact printing of patterned monolayers. On the basis of these results, we propose a mechanism for apoptotic cell extrusion: spontaneously formed topological defects in epithelia govern cell fate. This will be important in predicting extrusion hotspots and dynamics in vivo, with potential applications to tissue regeneration and the suppression of metastasis. Moreover, we anticipate that the analogy between the epithelium and active nematic liquid crystals will trigger further investigations of the link between cellular processes and the material properties of epithelia.

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Topological defects in epithelia govern cell death and extrusion.

Tumor invasion and metastasis are strongly regulated by biophysical interactions between tumor cells and the extracellular matrix (ECM). While the influence of ECM stiffness on cell migration, adhesion, and contractility has been extensively studied in 2D culture, extension of this concept to 3D cultures that more closely resemble tissue has proven challenging, because perturbations that change matrix stiffness often concurrently change cellular confinement. This coupling is particularly problematic given that matrix-imposed steric barriers can regulate invasion speed independent of mechanics. Here we introduce a matrix platform based on microfabrication of channels of defined wall stiffness and geometry that allows independent variation of ECM stiffness and channel width. For a given ECM stiffness, cells confined to narrow channels surprisingly migrate faster than cells in wide channels or on unconstrained 2D surfaces, which we attribute to increased polarization of cell-ECM traction forces. Confinement also enables cells to migrate increasingly rapidly as ECM stiffness rises, in contrast with the biphasic relationship observed on unconfined ECMs. Inhibition of nonmuscle myosin II dissipates this traction polarization and renders the relationship between migration speed and ECM stiffness comparatively insensitive to matrix confinement. We test these hypotheses in silico by devising a multiscale mathematical model that relates cellular force generation to ECM stiffness and geometry, which we show is capable of recapitulating key experimental trends. These studies represent a paradigm for investigating matrix regulation of invasion and demonstrate that matrix confinement alters the relationship between cell migration speed and ECM stiffness.

https://www.pnas.org/content/pnas/109/26/10334.full.pdf

Independent regulation of tumor cell migration by matrix stiffness and confinement

DiameterJ: A validated open source nanofiber diameter measurement tool.

We give a methodology-oriented perspective on directional image analysis and rotation-invariant processing. We review the state of the art in the field and make connections with recent mathematical developments in functional analysis and wavelet theory. We unify our perspective within a common framework using operators. The intent is to provide image-processing methods that can be deployed in algorithms that analyze biomedical images with improved rotation invariance and high directional sensitivity. We start our survey with classical methods such as directional-gradient and the structure tensor. Then, we discuss how these methods can be improved with respect to robustness, invariance to geometric transformations (with a particular interest in scaling), and computation cost. To address robustness against noise, we move forward to higher degrees of directional selectivity and discuss Hessian-based detection schemes. To present multiscale approaches, we explain the differences between Fourier filters, directional wavelets, curvelets, and shearlets. To reduce the computational cost, we address the problem of matching directional patterns by proposing steerable filters, where one might perform arbitrary rotations and optimizations without discretizing the orientation. We define the property of steerability and give an introduction to the design of steerable filters. We cover the spectrum from simple steerable filters through pyramid schemes up to steerable wavelets. We also present illustrations on the design of steerable wavelets and their application to pattern recognition.

/pdf/transforms-and-operators-for-directional-bioimage-analysis-a-cz4ketezr8.pdf

Transforms and Operators for Directional Bioimage Analysis: A Survey

Mechanical properties of the adventitia are largely determined by the organization of collagen fibers. Measurements on the waviness and orientation of collagen, particularly at the zero-stress state, are necessary to relate the structural organization of collagen to the mechanical response of the adventitia. Using the fluorescence collagen marker CNA38-OG488 and confocal laser scanning microscopy, we imaged collagen fibers in the adventitia of rabbit common carotid arteries ex vivo. The arteries were cut open along their longitudinal axes to get the zero-stress state. We used semi-manual and automatic techniques to measure parameters related to the waviness and orientation of fibers. Our results showed that the straightness parameter (defined as the ratio between the distances of endpoints of a fiber to its length) was distributed with a beta distribution (mean value 0.72, variance 0.028) and did not depend on the mean angle orientation of fibers. Local angular density distributions revealed four axially symmetric families of fibers with mean directions of 0°, 90°, 43° and −43°, with respect to the axial direction of the artery, and corresponding circular standard deviations of 40°, 47°, 37° and 37°. The distribution of local orientations was shifted to the circumferential direction when measured in arteries at the zero-load state (intact), as compared to arteries at the zero-stress state (cut-open). Information on collagen fiber waviness and orientation, such as obtained in this study, could be used to develop structural models of the adventitia, providing better means for analyzing and understanding the mechanical properties of vascular wall.

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Experimental investigation of collagen waviness and orientation in the arterial adventitia using confocal laser scanning microscopy.

Background: Structural constitutive models of vascular wall integrate information on composition and structural arrangements of tissue. In blood vessels, collagen fibres are arranged in coiled and wavy bundles and the individual collagen fibres have a deviation from their mean orientation. A complete structural constitutive model for vascular wall should incorporate both waviness and orientational distribution of fibres. We have previously developed a model, for passive properties of vascular wall, which considers the waviness of collagen fibres. However, to our knowledge there is no structural model of vascular wall which integrates both these features. Methods: In this study, we have suggested a structural strain energy function that incorporates not only the waviness but also the angular dispersion of fibres. We studied the effect of parameters related to the orientational distribution on macromechanical behaviour of tissue during inflation-extension tests. The model was further applied on experimental data from rabbit facial veins. Results: Our parametric study showed that the model is less sensitive to the orientational dispersion when fibres are mainly oriented circumferentially. The macromechanical response is less sensitive to changes in the mean orientation when fibres are more dispersed. The model accurately fitted the experimental data of veins, while not improving the quality of the fit compared to the model without dispersion. Our results showed that the orientational dispersion of collagen fibres could be compensated by a less abrupt and shifted to higher strain collagen engagement pattern. This should be considered when the model is fitted to experimental data and model parameters are used to study structural modifications of collagen fibre network in physiology and disease. Conclusions: The presented model incorporates structural features related to waviness and orientational distribution of collagen fibres and thus offers possibilities to better understand the relation between structure and function in the vascular wall. Also, the model can be used to further study mechanically induced collagen remodelling in vascular tissue in health and disease.

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Aristotelis Agianniotis

Papers

Experimental investigation of collagen waviness and orientation in the arterial adventitia using confocal laser scanning microscopy.

A structural constitutive model considering angular dispersion and waviness of collagen fibres of rabbit facial veins

Active axial stress in mouse aorta

Wall properties of the apolipoprotein E-deficient mouse aorta