Stretch-induced damage in endothelial monolayers.

Endothelial cells are constantly exposed to mechanical stimuli, of which mechanical stretch has shown various beneficial or deleterious effects depending on whether loads are within physiological or pathological levels, respectively. Vascular properties change with age, and on a cell-scale, senescence elicits changes in endothelial cell mechanical properties that together can impair its response to stretch. Here, high-rate uniaxial stretch experiments were performed to quantify and compare the stretch-induced damage of monolayers consisting of young, senescent, and aged endothelial populations. The aged and senescent phenotypes were more fragile to stretch-induced damage. Prominent damage was detected by immunofluorescence and scanning electron microscopy as intercellular and intracellular void formation. Damage increased proportionally to the applied level of deformation and, for the aged and senescent phenotype, induced significant detachment of cells at lower levels of stretch compared to the young counterpart. Based on the phenotypic difference in cell-substrate adhesion of senescent cells indicating more mature focal adhesions, a discrete network model of endothelial cells being stretched was developed. The model showed that the more affine deformation of senescent cells increased their intracellular energy, thus enhancing the tendency for cellular damage and impending detachment. Next to quantifying for the first-time critical levels of endothelial stretch, the present results indicate that young cells are more resilient to deformation and that the fragility of senescent cells may be associated with their stronger adhesion to the substrate.

Variations in fluid chemical potential induce fibroblast mechano-response in 3D hydrogels.

Mechanical deformation of skin creates variations in fluid chemical potential, leading to local changes in hydrostatic and osmotic pressure, whose effects on mechanobiology remain poorly understood. To study these effects, we investigate the specific influences of hydrostatic and osmotic pressure on primary human dermal fibroblasts in three-dimensional hydrogel culture models. Cyclic hydrostatic pressure and hyperosmotic stress enhanced the percentage of cells expressing the proliferation marker Ki67 in both collagen and PEG-based hydrogels. Osmotic pressure also activated the p38 MAPK stress response pathway and increased the expression of the osmoresponsive genes PRSS35 and NFAT5. When cells were cultured in two-dimension (2D), no change in proliferation was observed with either hydrostatic or osmotic pressure. Furthermore, basal, and osmotic pressure-induced expression of osmoresponsive genes differed in 2D culture versus 3D hydrogels, highlighting the role of dimensionality in skin cell mechanotransduction and stressing the importance of 3D tissue-like models that better replicate in vivo conditions. Overall, these results indicate that fluid chemical potential changes affect dermal fibroblast mechanobiology, which has implications for skin function and for tissue regeneration strategies.

Mechanical factors influence ß-catenin localization and barrier properties.

Mechanical forces are of major importance in regulating vascular homeostasis by influencing endothelial cell behavior and functions. Adherens junctions are critical sites for mechanotransduction in endothelial cells. ß-catenin, a component of adherens junctions and the canonical Wnt signaling pathway, plays a role in mechanoactivation. Evidence suggests that ß-catenin is involved in flow sensing and responds to tensional forces, impacting junction dynamics. The mechanoregulation of ß-catenin signaling is context-dependent, influenced by the type and duration of mechanical loads. In endothelial cells, ß-catenin's nuclear translocation and signaling are influenced by shear stress and strain, affecting endothelial permeability. The study investigates how shear stress, strain, and surface topography impact adherens junction dynamics, regulate ß-catenin localization, and influence endothelial barrier properties. Insight box Mechanical loads are potent regulators of endothelial functions through not completely elucidated mechanisms. Surface topography, wall shear stress and cyclic wall deformation contribute overlapping mechanical stimuli to which endothelial monolayer respond to adapt and maintain barrier functions. The use of custom developed flow chamber and bioreactor allows quantifying the response of mature human endothelial to well-defined wall shear stress and gradients of strain. Here, the mechanoregulation of ß-catenin by substrate topography, wall shear stress, and cyclic stretch is analyzed and linked to the monolayer control of endothelial permeability.

Biophysical Reviews' Topical issue: The 7th Nanoengineering for Mechanobiology Symposium 2024 Camogli, Genoa, Italy.

This Commentary describes an open call for submissions to the upcoming Biophysical Reviews' Issue Focus: The 7th Nanoengineering for Mechanobiology (Genova, Italy). The submission deadline is August 1st of 2024. Interested parties are requested to make contact with the Issue Focus editors prior to submission.

Discrete network models of endothelial cells and their interactions with the substrate.

Endothelial cell monolayers line the inner surfaces of blood and lymphatic vessels. They are continuously exposed to different mechanical loads, which may trigger mechanobiological signals and hence play a role in both physiological and pathological processes. Computer-based mechanical models of cells contribute to a better understanding of the relation between cell-scale loads and cues and the mechanical state of the hosting tissue. However, the confluency of the endothelial monolayer complicates these approaches since the intercellular cross-talk needs to be accounted for in addition to the cytoskeletal mechanics of the individual cells themselves. As a consequence, the computational approach must be able to efficiently model a large number of cells and their interaction. Here, we simulate cytoskeletal mechanics by means of molecular dynamics software, generally suitable to deal with large, locally interacting systems. Methods were developed to generate models of single cells and large monolayers with hundreds of cells. The single-cell model was considered for a comparison with experimental data. To this end, we simulated cell interactions with a continuous, deformable substrate, and computationally replicated multistep traction force microscopy experiments on endothelial cells. The results indicate that cell discrete network models are able to capture relevant features of the mechanical behaviour and are thus well-suited to investigate the mechanics of the large cytoskeletal network of individual cells and cell monolayers.

Protein Isolation from 3D Hydrogel Scaffolds.

Protein isolation is an essential tool in cell biology to characterize protein abundance under various experimental conditions. Several protocols exist, tailored to cell culture or tissue sections, and have been adapted to particular downstream analyses (e.g., western blotting or mass spectrometry). An increasing trend in bioengineering and cell biology is to use three-dimensional (3D) hydrogel-based scaffolds for cell culture. In principle, the same protocols can be used to extract protein from hydrogel-based cell and tissue constructs. However, in practice the yield and quality of the recovered protein pellet is often substantially lower when using standard protocols and requires tuning of multiple steps, including the selected lysis buffer and the scaffold homogenization strategy, as well as the methods for protein purification and reconstitution. We present here specific protocols tailored to common 3D hydrogels to help researchers using hydrogel-based 3D cell culture improve the quantity and quality of their extracted protein. We focus on three materials: protease-degradable PEG-based hydrogels, collagen hydrogels, and alginate hydrogels. We discuss how the protein extraction procedure can be adapted to the scaffold of interest (degradable or non-degradable gels), proteins of interests (soluble, matrix-bound, or phosphoproteins), and downstream biochemical assays (western blotting or mass spectrometry). With the growing interest in 3D cell culture, the protocols presented should be useful to many researchers in cell biology, protein science, biomaterials, and bioengineering communities. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolating proteins from PEG-based hydrogels Basic Protocol 2: Isolating proteins from collagen hydrogels Basic Protocol 3: Isolating proteins from alginate hydrogels Alternate Protocol: Isolating protein from alginate gels using EDTA to dissolve the gel Support Protocol: Isolating protein and RNA simultaneously from the same samples.

Radial matrix constraint influences tissue contraction and promotes maturation of bi-layered skin equivalents.

Human skin equivalents (HSEs) serve as important tools for mechanistic studies with human skin cells, drug discovery, pre-clinical applications in the field of tissue engineering and for skin transplantation on skin defects. Besides the cellular and extracellular matrix (ECM) components used for HSEs, physical constraints applied on the scaffold during HSEs maturation influence tissue organization, functionality, and homogeneity. In this study, we introduce a 3D-printed culture insert that exposes bi-layered HSEs to a static radial constraint through matrix adhesion. We examine the effect of various diameters of the ring-shaped culture insert on the HSE's characteristics and compare them to state-of-the-art unconstrained and planar constrained HSEs. We show that radial matrix constraint of HSEs regulates tissue contraction, promotes fibroblast and matrix organization that is similar to human skin in vivo and improves keratinocyte differentiation, epidermal stratification, and basement membrane formation depending on the culture insert diameter. Together, these data demonstrate that the degree of HSE's contraction is an important design consideration in skin tissue engineering. Therefore, this study can help to mimic various in vivo skin conditions and to increase the control of relevant tissue properties.

Endothelial damage inhibitor preserves the integrity of venous endothelial cells from patients undergoing coronary bypass surgery.

OBJECTIVES: Despite the success of coronary artery bypass graft (CABG) surgery using autologous saphenous vein grafts (SVGs), nearly 50% of patients experience vein graft disease within 10 years of surgery. One contributing factor to early vein graft disease is endothelial damage during short-term storage of SVGs in inappropriate solutions. Our aim was to evaluate the effects of a novel endothelial damage inhibitor (EDI) on SVGs from patients undergoing elective CABG surgery and on venous endothelial cells (VECs) derived from these SVGs. METHODS: SVGs from 11 patients participating in an ongoing clinical registry (NCT02922088) were included in this study, and incubated with both full electrolyte solution (FES) or EDI for 1 h and then examined histologically. In 8 of 11 patients, VECs were isolated from untreated grafts, incubated with both FES and EDI for 2 h under hypothermic stress conditions and then analysed for activation of an inflammatory phenotype, cell damage and cytotoxicity, as well as endothelial integrity and barrier function. RESULTS: The EDI was superior to FES in protecting the endothelium in SVGs (74 ± 8% versus 56 ± 8%, P < 0.001). Besides confirming that the EDI prevents apoptosis in SVG-derived VECs, we also showed that the EDI temporarily reduces adherens junctions in VECs while protecting focal adhesions compared to FES. CONCLUSIONS: The EDI protects the connectivity and function of the SVG endothelium. Our data suggest that the EDI can preserve focal adhesions in VECs during short-term storage after graft harvesting. This might explain the superiority of the EDI in maintaining most of the endothelium in venous CABG surgery conduits.

Multiscale mechanical analysis of the elastic modulus of skin.

The mechanical properties of the skin determine tissue function and regulate dermal cell behavior. Yet measuring these properties remains challenging, as evidenced by the large range of elastic moduli reported in the literature-from below one kPa to hundreds of MPa. Here, we reconcile these disparate results by dedicated experiments at both tissue and cellular length scales and by computational models considering the multiscale and multiphasic tissue structure. At the macroscopic tissue length scale, the collective behavior of the collagen fiber network under tension provides functional tissue stiffness, and its properties determine the corresponding elastic modulus (100-200 kPa). The compliant microscale environment (0.1-10 kPa), probed by atomic force microscopy, arises from the ground matrix without engaging the collagen fiber network. Our analysis indicates that indentation-based elasticity measurements, although probing tissue properties at the cell-relevant length scale, do not assess the deformation mechanisms activated by dermal cells when exerting traction forces on the extracellular matrix. Using dermal-equivalent collagen hydrogels, we demonstrate that indentation measurements of tissue stiffness do not correlate with the behavior of embedded dermal fibroblasts. These results provide a deeper understanding of tissue mechanics across length scales with important implications for skin mechanobiology and tissue engineering. STATEMENT OF SIGNIFICANCE: Measuring the mechanical properties of the skin is essential for understanding dermal cell mechanobiology and designing tissue-engineered skin substitutes. However, previous results reported for the elastic modulus of skin vary by six orders of magnitude. We show that two distinct deformation mechanisms, related to the tension-compression nonlinearity of the collagen fiber network, can explain the large variations in elastic moduli. Furthermore, we show that microscale indentation, which is frequently used to assess the stiffness perceived by cells, fails to engage the fiber network, and therefore cannot predict the behavior of dermal fibroblasts in stiffness-tunable fibrous hydrogels. This has important implications for how to measure and interpret the mechanical properties of soft tissues across length scales.

In-vitro investigation of endothelial monolayer retention on an inflow VAD cannula inside a beating heart phantom.

Ventricular assist devices (VADs) provide an alternative solution to heart transplantation for patients with end-stage heart failure. Insufficient hemocompatibility of VAD components can result in severe adverse events, such as thromboembolic stroke, and readmissions. To enhance VAD hemocompatibility, and avoid thrombus formation, surface modification techniques and endothelialization strategies are employed. In this work, a free form patterning topography is selected to facilitate the endothelialization of the outer surface of the inflow cannula (IC) of a commercial VAD. An endothelialization protocol for convoluted surfaces such as the IC is produced, and the retainment of the endothelial cell (EC) monolayer is evaluated. To allow this evaluation, a dedicated experimental setup is developed to simulate realistic flow phenomena inside an artificial, beating heart phantom with a VAD implanted on its apex. The procedural steps of mounting the system result to the impairment of the EC monolayer, which is further compromised by the developed flow and pressure conditions, as well as by the contact with the moving inner structures of the heart phantom. Importantly, the EC monolayer is better maintained in the lower part of the IC, which is more susceptible to thrombus formation and may therefore aid in minimizing the hemocompatibility related adverse events after the VAD implantation.

DYRK1B inhibition exerts senolytic effects on endothelial cells and rescues endothelial dysfunctions.

Aging is the major risk factor for chronic disease development. Cellular senescence is a key mechanism that triggers or contributes to age-related phenotypes and pathologies. The endothelium, a single layer of cells lining the inner surface of a blood vessel, is a critical interface between blood and all tissues. Many studies report a link between endothelial cell senescence, inflammation, and diabetic vascular diseases. Here we identify, using combined advanced AI and machine learning, the Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1B (DYRK1B) protein as a possible senolytic target for senescent endothelial cells. We demonstrate that upon induction of senescence in vitro DYRK1B expression is increased in endothelial cells and localized at adherens junctions where it impairs their proper organization and functions. DYRK1B knock-down or inhibition restores endothelial barrier properties and collective behavior. DYRK1B is therefore a possible target to counteract diabetes-associated vascular diseases linked to endothelial cell senescence.

Accelerated epithelial layer healing induced by tactile anisotropy in surface topography.

Mammalian cells respond to tactile cues from topographic elements presented by the substrate. Among these, anisotropic features distributed in an ordered manner give directionality. In the extracellular matrix, this ordering is embedded in a noisy environment altering the contact guidance effect. To date, it is unclear how cells respond to topographical signals in a noisy environment. Here, using rationally designed substrates, we report morphotaxis, a guidance mechanism enabling fibroblasts and epithelial cells to move along gradients of topographic order distortion. Isolated cells and cell ensembles perform morphotaxis in response to gradients of different strength and directionality, with mature epithelia integrating variations of topographic order over hundreds of micrometers. The level of topographic order controls cell cycle progression, locally delaying or promoting cell proliferation. In mature epithelia, the combination of morphotaxis and noise-dependent distributed proliferation provides a strategy to enhance wound healing as confirmed by a mathematical model capturing key elements of the process.

Patient's dermal fibroblasts as disease markers for visceral myopathy.

Visceral myopathy (VSCM) is a rare genetic disease, orphan of pharmacological therapy. VSCM diagnosis is not always straightforward due to symptomatology similarities with mitochondrial or neuronal forms of intestinal pseudo-obstruction. The most prevalent form of VSCM is associates with variants in the gene ACTG2, encoding the protein gamma-2 actin. Overall, VSCM is a mechano-biological disorder, in which different genetic variants lead to similar alterations to the contractile phenotype of enteric smooth muscles, resulting in the emergence of life-threatening symptoms. In this work we analyzed the morpho-mechanical phenotype of human dermal fibroblasts from patients affected with VSCM, demonstrating that they retain a clear signature of the disease when compared with different controls. We evaluated several biophysical traits of fibroblasts, and we show that a measure of cellular traction forces can be used as a non-specific biomarker of the disease. We propose that a simple assay based on traction forces could be designed to provide a valuable support for clinical decision or pre-clinical research.

Control of hydrostatic pressure and osmotic stress in 3D cell culture for mechanobiological studies.

Hydrostatic pressure (HP) and osmotic stress (OS) play an important role in various biological processes, such as cell proliferation and differentiation. In contrast to canonical mechanical signals transmitted through the anchoring points of the cells with the extracellular matrix, the physical and molecular mechanisms that transduce HP and OS into cellular functions remain elusive. Three-dimensional cell cultures show great promise to replicate physiologically relevant signals in well-defined host bioreactors with the goal of shedding light on hidden aspects of the mechanobiology of HP and OS. This review starts by introducing prevalent mechanisms for the generation of HP and OS signals in biological tissues that are subject to pathophysiological mechanical loading. We then revisit various mechanisms in the mechanotransduction of HP and OS, and describe the current state of the art in bioreactors and biomaterials for the control of the corresponding physical signals.

Anisotropic topographies restore endothelial monolayer integrity and promote the proliferation of senescent endothelial cells.

Thrombogenicity remains a major issue in cardiovascular implants (CVIs). Complete surficial coverage of CVIs by a monolayer of endothelial cells (ECs) prior to implantation represents a promising strategy but is hampered by the overall logistical complexity and the high number of cells required. Consequently, extensive cell expansion is necessary, which may eventually lead to replicative senescence. Considering that micro-structured surfaces with anisotropic topography may promote endothelialization, we investigated the impact of gratings on the biomechanical properties and the replicative capacity of senescent ECs. After cultivation on gridded surfaces, the cells showed significant improvements in terms of adherens junction integrity, cell elongation, and orientation of the actin filaments, as well as enhanced yes-associated protein nuclear translocation and cell proliferation. Our data therefore suggest that micro-structured surfaces with anisotropic topographies may improve long-term endothelialization of CVIs.

A novel bistable device to study mechanosensitive cell responses to instantaneous stretch.

The behavior of cells and tissues in vivo is determined by the integration of multiple biochemical and mechanical signals. Of the mechanical signals, stretch has been studied for decades and shown to contribute to pathophysiological processes. Several different stretch devices have been developed for in vitro investigations of cell stretch. In this work, we describe a new 3D-printed uniaxial stretching device for studying cell response to rapid deformation. The device is a bistable compliant mechanism holding two equilibrium states-an unstretched and stretched configuration-without the need of an external actuator. Furthermore, it allows multiple simultaneous measurements of different levels of stretch on a single substrate and is compatible with standard immunofluorescence imaging of fixed cells as well as live-cell imaging. To demonstrate the effectiveness of the device to stretch cells, a test case using aligned myotubes is presented. Leveraging material area changes associated with deformation of the substrate, changes in nuclei density provided evidence of affine deformation between cells and substrate. Furthermore, intranuclear deformations were also assessed and shown to deform non-affinely. As a proof-of-principle of the use of the device for mechanobiological studies, we uniaxially stretched aligned healthy and dystrophic myotubes that displayed different passive mechanical responses, consistent with previous literature in the field. We also identified a new feature in the mechanoresponse of dystrophic myotubes, which is of potential interest for identifying the diseased cells based on a quick mechanical readout. While some applications of the device for elucidating passive mechanical responses are demonstrated, the simplicity of the device allows it to be potentially used for other modes of deformation with little modifications.

The path to a hemocompatible cardiovascular implant: Advances and challenges of current endothelialization strategies.

Cardiovascular (CV) implants are still associated with thrombogenicity due to insufficient hemocompatibility. Endothelialization of their luminal surface is a promising strategy to increase their hemocompatibility. In this review, we provide a collection of research studies and review articles aiming to summarize the recent efforts on surface modifications of CV implants, including stents, grafts, valves, and ventricular assist devises. We focus in particular on the implementation of micrometer or nanoscale surface modifications, physical characteristics of known biomaterials (such as wetness and stiffness), and surface morphological features (such as gratings, fibers, pores, and pits). We also review how biomechanical signals originating from the endothelial cell for surface interaction can be directed by topography engineering approaches toward the survival of the endothelium and its long-term adaptation. Finally, we summarize the regulatory and economic challenges that may prevent clinical implementation of endothelialized CV implants.

Bistability of Dielectrically Anisotropic Nematic Crystals and the Adaptation of Endothelial Collectives to Stress Fields.

Endothelial monolayers physiologically adapt to flow and flow-induced wall shear stress, attaining ordered configurations in which elongation, orientation, and polarization are coherently organized over many cells. Here, with the flow direction unchanged, a peculiar bi-stable (along the flow direction or perpendicular to it) cell alignment is observed, emerging as a function of the flow intensity alone, while cell polarization is purely instructed by flow directionality. Driven by the experimental findings, the parallelism between endothelia is delineated under a flow field and the transition of dual-frequency nematic liquid crystals under an external oscillatory electric field. The resulting physical model reproduces the two stable configurations and the energy landscape of the corresponding system transitions. In addition, it reveals the existence of a disordered, metastable state emerging upon system perturbation. This intermediate state, experimentally demonstrated in endothelial monolayers, is shown to expose the cellular system to a weakening of cell-to-cell junctions to the detriment of the monolayer integrity. The flow-adaptation of monolayers composed of healthy and senescent endothelia is successfully predicted by the model with adjustable nematic parameters. These results may help to understand the maladaptive response of in vivo endothelial tissues to disturbed hemodynamics and the progressive functional decay of senescent endothelia.

A dual role of YAP in driving TGFß-mediated endothelial-to-mesenchymal transition.

Endothelial-to-mesenchymal transition (EndMT) is the biological process through which endothelial cells transdifferentiate into mesenchymal cells. During embryo development, EndMT regulates endocardial cushion formation via TGFß/BMP signaling. In adults, EndMT is mainly activated during pathological conditions. Hence, it is necessary to characterize molecular regulators cooperating with TGFß signaling in driving EndMT, to identify potential novel therapeutic targets to treat these pathologies. Here, we studied YAP, a transcriptional co-regulator involved in several biological processes, including epithelial-to-mesenchymal transition (EMT). As EndMT is the endothelial-specific form of EMT, and YAP (herein referring to YAP1) and TGFß signaling cross-talk in other contexts, we hypothesized that YAP contributes to EndMT by modulating TGFß signaling. We demonstrate that YAP is required to trigger TGFß-induced EndMT response, specifically contributing to SMAD3-driven EndMT early gene transcription. We provide novel evidence that YAP acts as SMAD3 transcriptional co-factor and prevents GSK3ß-mediated SMAD3 phosphorylation, thus protecting SMAD3 from degradation. YAP is therefore emerging as a possible candidate target to inhibit pathological TGFß-induced EndMT at early stages.

A ligand-insensitive UNC5B splicing isoform regulates angiogenesis by promoting apoptosis.

The Netrin-1 receptor UNC5B is an axon guidance regulator that is also expressed in endothelial cells (ECs), where it finely controls developmental and tumor angiogenesis. In the absence of Netrin-1, UNC5B induces apoptosis that is blocked upon Netrin-1 binding. Here, we identify an UNC5B splicing isoform (called UNC5B-Δ8) expressed exclusively by ECs and generated through exon skipping by NOVA2, an alternative splicing factor regulating vascular development. We show that UNC5B-Δ8 is a constitutively pro-apoptotic splicing isoform insensitive to Netrin-1 and required for specific blood vessel development in an apoptosis-dependent manner. Like NOVA2, UNC5B-Δ8 is aberrantly expressed in colon cancer vasculature where its expression correlates with tumor angiogenesis and poor patient outcome. Collectively, our data identify a mechanism controlling UNC5B's necessary apoptotic function in ECs and suggest that the NOVA2/UNC5B circuit represents a post-transcriptional pathway regulating angiogenesis.