Experimental investigation of collagen waviness and orientation in the arterial adventitia using confocal laser scanning microscopy.
01 Mar 2012-Biomechanics and Modeling in Mechanobiology (Springer Verlag)-Vol. 11, Iss: 3, pp 461-473
TL;DR: Information on collagen fiber waviness and orientation could be used to develop structural models of the adventitia, providing better means for analyzing and understanding the mechanical properties of vascular wall.
Abstract: 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.
Citations
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TL;DR: Piezo1 channels are shown as sensors of frictional force (shear stress) and determinants of vascular structure in both development and adult physiology and the data suggest that Piezo 1 channels function as pivotal integrators in vascular biology.
Abstract: 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.
710 citations
TL;DR: A mechanism for apoptotic cell extrusion is proposed: spontaneously formed topological defects in epithelia govern cell fate, and the ability to control extrusion hotspots by geometrically inducing defects through microcontact printing of patterned monolayers is demonstrated.
Abstract: 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.
556 citations
TL;DR: A matrix platform based on microfabrication of channels of defined wall stiffness and geometry that allows independent variation of ECM stiffness and channel width is introduced and it is demonstrated that matrix confinement alters the relationship between cell migration speed andECM stiffness.
Abstract: 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.
483 citations
TL;DR: The goal of this study was to create a user friendly ImageJ/FIJI plugin that would analyze SEM micrographs of nanofibers to determine nanofiber diameter on a desktop computer within 60 s.
396 citations
Cites methods from "Experimental investigation of colla..."
...Several other labs have developed tools to assess nanofiber diameter using edge detection algorithms [40], Radon Transforms [39], or principal component analysis [41] however, these tools were never released to the community and were therefore never validated....
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TL;DR: 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, and address the problem of matching directional patterns by proposing steerable filters.
Abstract: 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.
333 citations
References
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01 May 1984
2,493 citations
"Experimental investigation of colla..." refers background in this paper
...The closer the probability plot of data to this line, the better the data follow that particular distribution (Chambers et al. 1983)....
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01 Jan 1983
TL;DR: This paper presents a meta-modelling framework for developing and assessing regression models for multivariate and multi-dimensional data distributions and describes the distribution of a set of data.
Abstract: This book present graphical methods for analysing data. Some methods are new and some are old, some require a computer and others only paper and pencil; but they are all powerful data analysis tools. In many situations, a set of data � even a large set- can be adequately analysed through graphical methods alone. In most other situations, a few well-chosen graphical displays can significantly enhance numerical statistical analyses.
1,763 citations
"Experimental investigation of colla..." refers background in this paper
...The closer the probability plot of data to this line, the better the data follow that particular distribution (Chambers et al. 1983)....
[...]
TL;DR: The design and validation of a semiautomatic neurite tracing technique for accurate and reproducible segmentation and quantification of neuronal processes are described.
Abstract: Background: For the investigation of the molecular mechanisms involved in neurite outgrowth and differentiation, accurate and reproducible segmentation and quantification of neuronal processes are a prerequisite. To facilitate this task, we developed a semiautomatic neurite tracing technique. This article describes the design and validation of the technique. Methods: The technique was compared to fully manual delineation. Four observers repeatedly traced selected neurites in 20 fluorescence microscopy images of cells in culture, using both methods. Accuracy and reproducibility were determined by comparing the tracings to high-resolution reference tracings, using two error measures. Labor intensiveness was measured in numbers of mouse clicks required. The significance of the results was determined by a Student t-test and by analysis of variance. Results: Both methods slightly underestimated the true neurite length, but the differences were not unanimously significant. The average deviation from the true neurite centerline was a factor 2.6 smaller with the developed technique compared to fully manual tracing. Intraobserver variability in the respective measures was reduced by a factor 6.0 and 23.2. Interobserver variability was reduced by a factor 2.4 and 8.8, respectively, and labor intensiveness by a factor 3.3. Conclusions: Providing similar accuracy in measuring neurite length, significantly improved accuracy in neurite centerline extraction, and significantly improved reproducibility and reduced labor intensiveness, the developed technique may replace fully manual tracing methods.
1,417 citations
"Experimental investigation of colla..." refers methods in this paper
...These parameters were measured using NeuronJ, an ImageJ plug-in originally developed for Neurite tracing and analysis ( Meijering et al. 2004 )....
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...These parameters were measured using NeuronJ, an ImageJ plug-in originally developed for Neurite tracing and analysis (Meijering et al. 2004)....
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TL;DR: Recent studies of the three-dimensional in vivo structures of well-ordered protein assemblies, such as collagen, microtubules and muscle myosin, are beginning to establish SHIM as a nondestructive imaging modality that holds promise for both basic research and clinical pathology.
Abstract: Although the nonlinear optical effect known as second-harmonic generation (SHG) has been recognized since the earliest days of laser physics and was demonstrated through a microscope over 25 years ago, only in the past few years has it begun to emerge as a viable microscope imaging contrast mechanism for visualization of cell and tissue structure and function. Only small modifications are required to equip a standard laser-scanning two-photon microscope for second-harmonic imaging microscopy (SHIM). Recent studies of the three-dimensional in vivo structures of well-ordered protein assemblies, such as collagen, microtubules and muscle myosin, are beginning to establish SHIM as a nondestructive imaging modality that holds promise for both basic research and clinical pathology. Thus far the best signals have been obtained in a transmitted light geometry that precludes in vivo measurements on large living animals. This drawback may be addressed through improvements in the collection of SHG signals via an epi-illumination microscope configuration. In addition, SHG signals from certain membrane-bound dyes have been shown to be highly sensitive to membrane potential. Although this indicates that SHIM may become a valuable tool for probing cell physiology, the small signal size would limit the number of photons that could be collected during the course of a fast action potential. Better dyes and optimized microscope optics could ultimately lead to the imaging of neuronal electrical activity with SHIM.
1,291 citations
"Experimental investigation of colla..." refers background in this paper
...They observed that the SHG in backward geometry was much weaker than the fluorescent signal of the probe....
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...…labeling agents or fixative as a result of its intrinsic properties such as bifringence under polarized light microscopy (Finlay et al. 1995; Hilbert et al. 1996), auto-fluorescence (Voytik-Harbin et al. 2001), and second harmonic generation (SHG) (Campagnola and Loew 2003; Boulesteix et al. 2006)....
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...In a recent study, Boerboom et al. (2007) compared the imaging of collagen with CNA35 probe and backward geometry SHG signal....
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...2001), and second harmonic generation (SHG) (Campagnola and Loew 2003; Boulesteix et al. 2006)....
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...As for the SHG, strong forward scattered SHG does enable the visualization of collagen in tissues; however, depending on tissue properties, forward SHG is not always feasible....
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