Actomyosin-dependent invasion of endothelial sprouts in collagen.

During sprouting angiogenesis-the growth of blood vessels from the existing vasculature-endothelial cells (ECs) adopt an elongated invasive form and exert forces at cell-cell and cell-matrix interaction sites. These cell shape changes and cellular tractions require extensive reorganizations of the actomyosin network. However, the respective roles of actin and myosin for endothelial sprouting are not fully elucidated. In this study, we further investigate these roles by treating 2D-migrating and 3D-sprouting ECs with chemical compounds targeting either myosin or actin. These treatments affected the endothelial cytoskeleton drastically and reduced the invasive response in a compound-specific manner; pointing toward a tight control of the actin and myosin activity during sprouting. Clusters in the data further illustrate that endothelial sprout morphology is sensitive to the in vitro model mechanical microenvironment and directs future research toward mechanical substrate guidance as a strategy for promoting engineered tissue vascularization. In summary, our results add to a growing corpus of research highlighting a key role of the cytoskeleton for sprouting angiogenesis.

Matrix deformations around angiogenic sprouts correlate to sprout dynamics and suggest pulling activity.

Angiogenesis is the formation of new blood vessels from the pre-existing vasculature. It is essential for normal tissue growth and regeneration, and also plays a key role in many diseases [Carmeliet in Nat Med 9:653-660, 2003]. Cytoskeletal components have been shown to be important for angiogenic sprout initiation and maintenance [Kniazeva and Putnam in Am J Physiol 297:C179-C187, 2009] as well as endothelial cell shape control during invasion [Elliott et al. in Nat Cell Biol 17:137-147, 2015]. The exact nature of cytoskeleton-mediated forces for sprout initiation and progression, however, remains poorly understood. Questions on the importance of tip cell pulling versus stalk cell pushing are to a large extent unanswered, which among others has to do with the difficulty of quantifying and resolving those forces in time and space. We developed methods based on time-lapse confocal microscopy and image processing-further termed 4D displacement microscopy-to acquire detailed, spatially and temporally resolved extracellular matrix (ECM) deformations, indicative of cell-ECM mechanical interactions around invading sprouts. We demonstrate that matrix deformations dependent on actin-mediated force generation are spatio-temporally correlated with sprout morphological dynamics. Furthermore, sprout tips were found to exert radially pulling forces on the extracellular matrix, which were quantified by means of a computational model of collagen ECM mechanics. Protrusions from extending sprouts mostly increase their pulling forces, while retracting protrusions mainly reduce their pulling forces. Displacement microscopy analysis further unveiled a characteristic dipole-like deformation pattern along the sprout direction that was consistent among seemingly very different sprout shapes-with oppositely oriented displacements at sprout tip versus sprout base and a transition zone of negligible displacements in between. These results demonstrate that sprout-ECM interactions are dominated by pulling forces and underline the key role of tip cell pulling for sprouting angiogenesis.

Fast quantitative time lapse displacement imaging of endothelial cell invasion.

In order to unravel rapid mechano-chemical feedback mechanisms in sprouting angiogenesis, we combine selective plane illumination microscopy (SPIM) and tailored image registration algorithms - further referred to as SPIM-based displacement microscopy - with an in vitro model of angiogenesis. SPIM successfully tackles the problem of imaging large volumes while upholding the spatial resolution required for the analysis of matrix displacements at a subcellular level. Applied to in vitro angiogenic sprouts, this unique methodological combination relates subcellular activity - minute to second time scale growing and retracting of protrusions - of a multicellular systems to the surrounding matrix deformations with an exceptional temporal resolution of 1 minute for a stack with multiple sprouts simultaneously or every 4 seconds for a single sprout, which is 20 times faster than with a conventional confocal setup. Our study reveals collective but non-synchronised, non-continuous activity of adjacent sprouting cells along with correlations between matrix deformations and protrusion dynamics.

Spatiotemporal Analyses of Cellular Tractions Describe Subcellular Effect of Substrate Stiffness and Coating.

Cells interplay with their environment through mechanical and chemical interactions. To characterize this interplay, endothelial cells were cultured on polyacrylamide hydrogels of varying stiffness, coated with either fibronectin or collagen. We developed a novel analysis technique, complementary to traction force microscopy, to characterize the spatiotemporal evolution of cellular tractions: We identified subpopulations of tractions, termed traction foci, and tracked their magnitude and lifetime. Each focus consists of tractions associated with a local single peak of maximal traction. Individual foci were spread over a larger area in cells cultured on collagen relative to those on fibronectin and exerted higher tractions on stiffer hydrogels. We found that the trends with which forces increased with increasing hydrogel stiffness were different for foci and whole-cell measurements. These differences were explained by the number of foci and their average strength. While on fibronectin multiple short-lived weak foci contributed up to 30% to the total traction on hydrogels with intermediate stiffness, short-lived foci in such a number were not observed on collagen despite the higher tractions. Our approach allows for the use of existing traction force microscopy data to gain insight at the subcellular scale without molecular probes or spatial constraining of cellular tractions.

Combustion-derived particles inhibit in vitro human lung fibroblast-mediated matrix remodeling.

BACKGROUND: The continuously growing human exposure to combustion-derived particles (CDPs) drives in depth investigation of the involved complex toxicological mechanisms of those particles. The current study evaluated the hypothesis that CDPs could affect cell-induced remodeling of the extracellular matrix due to their underlying toxicological mechanisms. The effects of two ultrafine and one fine form of CDPs on human lung fibroblasts (MRC-5 cell line) were investigated, both in 2D cell culture and in 3D collagen type I hydrogels. A multi-parametric analysis was employed. RESULTS: In vitro dynamic 3D analysis of collagen matrices showed that matrix displacement fields induced by human lung fibroblasts are disturbed when exposed to carbonaceous particles, resulting in inhibition of matrix remodeling. In depth analysis using general toxicological assays revealed that a plausible explanation comprises a cascade of numerous detrimental effects evoked by the carbon particles, including oxidative stress, mitochondrial damage and energy storage depletion. Also, ultrafine particles revealed stronger toxicological and inhibitory effects compared to their larger counterparts. The inhibitory effects can be almost fully restored when treating the impaired cells with antioxidants like vitamin C. CONCLUSIONS: The unraveled in vitro pathway, by which ultrafine particles alter the fibroblasts' vital role of matrix remodeling, extends our knowledge about the contribution of these biologically active particles in impaired lung tissue repair mechanisms, and development and exacerbation of chronic lung diseases. The new insights may even pave the way to precautionary actions. The results provide justification for toxicological assessments to include mechanism-linked assays besides the traditional in vitro toxicological screening assays.

Full L1-regularized Traction Force Microscopy over whole cells.

BACKGROUND: Traction Force Microscopy (TFM) is a widespread technique to estimate the tractions that cells exert on the surrounding substrate. To recover the tractions, it is necessary to solve an inverse problem, which is ill-posed and needs regularization to make the solution stable. The typical regularization scheme is given by the minimization of a cost functional, which is divided in two terms: the error present in the data or data fidelity term; and the regularization or penalty term. The classical approach is to use zero-order Tikhonov or L2-regularization, which uses the L2-norm for both terms in the cost function. Recently, some studies have demonstrated an improved performance using L1-regularization (L1-norm in the penalty term) related to an increase in the spatial resolution and sensitivity of the recovered traction field. In this manuscript, we present a comparison between the previous two regularization schemes (relying in the L2-norm for the data fidelity term) and the full L1-regularization (using the L1-norm for both terms in the cost function) for synthetic and real data. RESULTS: Our results reveal that L1-regularizations give an improved spatial resolution (more important for full L1-regularization) and a reduction in the background noise with respect to the classical zero-order Tikhonov regularization. In addition, we present an approximation, which makes feasible the recovery of cellular tractions over whole cells on typical full-size microscope images when working in the spatial domain. CONCLUSIONS: The proposed full L1-regularization improves the sensitivity to recover small stress footprints. Moreover, the proposed method has been validated to work on full-field microscopy images of real cells, what certainly demonstrates it is a promising tool for biological applications.

3D full-field quantification of cell-induced large deformations in fibrillar biomaterials by combining non-rigid image registration with label-free second harmonic generation.

To advance our current understanding of cell-matrix mechanics and its importance for biomaterials development, advanced three-dimensional (3D) measurement techniques are necessary. Cell-induced deformations of the surrounding matrix are commonly derived from the displacement of embedded fiducial markers, as part of traction force microscopy (TFM) procedures. However, these fluorescent markers may alter the mechanical properties of the matrix or can be taken up by the embedded cells, and therefore influence cellular behavior and fate. In addition, the currently developed methods for calculating cell-induced deformations are generally limited to relatively small deformations, with displacement magnitudes and strains typically of the order of a few microns and less than 10% respectively. Yet, large, complex deformation fields can be expected from cells exerting tractions in fibrillar biomaterials, like collagen. To circumvent these hurdles, we present a technique for the 3D full-field quantification of large cell-generated deformations in collagen, without the need of fiducial markers. We applied non-rigid, Free Form Deformation (FFD)-based image registration to compute full-field displacements induced by MRC-5 human lung fibroblasts in a collagen type I hydrogel by solely relying on second harmonic generation (SHG) from the collagen fibrils. By executing comparative experiments, we show that comparable displacement fields can be derived from both fibrils and fluorescent beads. SHG-based fibril imaging can circumvent all described disadvantages of using fiducial markers. This approach allows measuring 3D full-field deformations under large displacement (of the order of 10 µm) and strain regimes (up to 40%). As such, it holds great promise for the study of large cell-induced deformations as an inherent component of cell-biomaterial interactions and cell-mediated biomaterial remodeling.

Biomechanical Characterization of Ascending Aortic Aneurysms.

Ascending thoracic aortic aneurysms (ATAAs) are a silent disease, ultimately leading to dissection or rupture of the arterial wall. There is a growing consensus that diameter information is insufficient to assess rupture risk, whereas wall stress and strength provide a more reliable estimate. The latter parameters cannot be measured directly and must be inferred through biomechanical assessment, requiring a thorough knowledge of the mechanical behaviour of the tissue. However, for healthy and aneurysmal ascending aortic tissues, this knowledge remains scarce. This study provides the geometrical and mechanical properties of the ATAA of six patients with unprecedented detail. Prior to their ATAA repair, pressure and diameter were acquired non-invasively, from which the distensibility coefficient, pressure-strain modulus and wall stress were calculated. Uniaxial tensile tests on the resected tissue yielded ultimate stress and stretch values. Parameters for the Holzapfel-Gasser-Ogden material model were estimated based on the pre-operative pressure-diameter data and the post-operative stress-stretch curves from planar biaxial tensile tests. Our results confirmed that mechanical or geometrical information alone cannot provide sufficient rupture risk estimation. The ratio of physiological to ultimate wall stress seems a more promising parameter. However, wall stress estimation suffers from uncertainties in wall thickness measurement, for which our results show large variability, between patients but also between measurement methods. Our results also show a large strength variability, a value which cannot be measured non-invasively. Future work should therefore be directed towards improved accuracy of wall thickness estimation, but also towards the large-scale collection of ATAA wall strength data.

Free Form Deformation-Based Image Registration Improves Accuracy of Traction Force Microscopy.

Traction Force Microscopy (TFM) is a widespread method used to recover cellular tractions from the deformation that they cause in their surrounding substrate. Particle Image Velocimetry (PIV) is commonly used to quantify the substrate's deformations, due to its simplicity and efficiency. However, PIV relies on a block-matching scheme that easily underestimates the deformations. This is especially relevant in the case of large, locally non-uniform deformations as those usually found in the vicinity of a cell's adhesions to the substrate. To overcome these limitations, we formulate the calculation of the deformation of the substrate in TFM as a non-rigid image registration process that warps the image of the unstressed material to match the image of the stressed one. In particular, we propose to use a B-spline -based Free Form Deformation (FFD) algorithm that uses a connected deformable mesh to model a wide range of flexible deformations caused by cellular tractions. Our FFD approach is validated in 3D fields using synthetic (simulated) data as well as with experimental data obtained using isolated endothelial cells lying on a deformable, polyacrylamide substrate. Our results show that FFD outperforms PIV providing a deformation field that allows a better recovery of the magnitude and orientation of tractions. Together, these results demonstrate the added value of the FFD algorithm for improving the accuracy of traction recovery.

Image analysis for the quantitative comparison of stress fibers and focal adhesions.

Actin stress fibers (SFs) detect and transmit forces to the extracellular matrix through focal adhesions (FAs), and molecules in this pathway determine cellular behavior. Here, we designed two different computational tools to quantify actin SFs and the distribution of actin cytoskeletal proteins within a normalized cellular morphology. Moreover, a systematic cell response comparison between the control cells and those with impaired actin cytoskeleton polymerization was performed to demonstrate the reliability of the tools. Indeed, a variety of proteins that were present within the string beginning at the focal adhesions (vinculin) up to the actin SFs contraction (non-muscle myosin II (NMMII)) were analyzed. Finally, the software used allows for the quantification of the SFs based on the relative positions of FAs. Therefore, it provides a better insight into the cell mechanics and broadens the knowledge of the nature of SFs.