Notch in mechanotransduction - from molecular mechanosensitivity to tissue mechanostasis.

Tissue development and homeostasis are controlled by mechanical cues. Perturbation of the mechanical equilibrium triggers restoration of mechanostasis through changes in cell behavior, while defects in these restorative mechanisms lead to mechanopathologies, for example, osteoporosis, myopathies, fibrosis or cardiovascular disease. Therefore, sensing mechanical cues and integrating them with the biomolecular cell fate machinery is essential for the maintenance of health. The Notch signaling pathway regulates cell and tissue fate in nearly all tissues. Notch activation is directly and indirectly mechanosensitive, and regulation of Notch signaling, and consequently cell fate, is integral to the cellular response to mechanical cues. Fully understanding the dynamic relationship between molecular signaling, tissue mechanics and tissue remodeling is challenging. To address this challenge, engineered microtissues and computational models play an increasingly large role. In this Review, we propose that Notch takes on the role of a 'mechanostat', maintaining the mechanical equilibrium of tissues. We discuss the reciprocal role of Notch in the regulation of tissue mechanics, with an emphasis on cardiovascular tissues, and the potential of computational and engineering approaches to unravel the complex dynamic relationship between mechanics and signaling in the maintenance of cell and tissue mechanostasis.

OpenABM-Covid19-An agent-based model for non-pharmaceutical interventions against COVID-19 including contact tracing.

SARS-CoV-2 has spread across the world, causing high mortality and unprecedented restrictions on social and economic activity. Policymakers are assessing how best to navigate through the ongoing epidemic, with computational models being used to predict the spread of infection and assess the impact of public health measures. Here, we present OpenABM-Covid19: an agent-based simulation of the epidemic including detailed age-stratification and realistic social networks. By default the model is parameterised to UK demographics and calibrated to the UK epidemic, however, it can easily be re-parameterised for other countries. OpenABM-Covid19 can evaluate non-pharmaceutical interventions, including both manual and digital contact tracing, and vaccination programmes. It can simulate a population of 1 million people in seconds per day, allowing parameter sweeps and formal statistical model-based inference. The code is open-source and has been developed by teams both inside and outside academia, with an emphasis on formal testing, documentation, modularity and transparency. A key feature of OpenABM-Covid19 are its Python and R interfaces, which has allowed scientists and policymakers to simulate dynamic packages of interventions and help compare options to suppress the COVID-19 epidemic.

Prediction of Cell Alignment on Cyclically Strained Grooved Substrates.

Cells respond to both mechanical and topographical stimuli by reorienting and reorganizing their cytoskeleton. Under certain conditions, such as for cells on cyclically stretched grooved substrates, the effects of these stimuli can be antagonistic. The biophysical processes that lead to the cellular reorientation resulting from such a competition are not clear yet. In this study, we hypothesized that mechanical cues and the diffusion of the intracellular signal produced by focal adhesions are determinants of the final cellular alignment. This hypothesis was investigated by means of a computational model, with the aim to simulate the (re)orientation of cells cultured on cyclically stretched grooved substrates. The computational results qualitatively agree with previous experimental studies, thereby supporting our hypothesis. Furthermore, cellular behavior resulting from experimental conditions different from the ones reported in the literature was simulated, which can contribute to the development of new experimental designs.

Steering cell orientation through light-based spatiotemporal modulation of the mechanical environment.

The anisotropic organization of cells and the extracellular matrix (ECM) is essential for the physiological function of numerous biological tissues, including the myocardium. This organization changes gradually in space and time, during disease progression such as myocardial infarction. The role of mechanical stimuli has been demonstrated to be essential in obtaining, maintaining and de-railing this organization, but the underlying mechanisms are scarcely known. To enable the study of the mechanobiological mechanisms involved,in vitrotechniques able to spatiotemporally control the multiscale tissue mechanical environment are thus necessary. Here, by using light-sensitive materials combined with light-illumination techniques, we fabricated 2D and 3Din vitromodel systems exposing cells to multiscale, spatiotemporally resolved stiffness anisotropies. Specifically, spatial stiffness anisotropies spanning from micron-sized (cellular) to millimeter-sized (tissue) were achieved. Moreover, the light-sensitive materials allowed to introduce the stiffness anisotropies at defined timepoints (hours) after cell seeding, facilitating the study of their temporal effects on cell and tissue orientation. The systems were tested using cardiac fibroblasts (cFBs), which are known to be crucial for the remodeling of anisotropic cardiac tissue. We observed that 2D stiffness micropatterns induced cFBs anisotropic alignment, independent of the stimulus timing, but dependent on the micropattern spacing. cFBs exhibited organized alignment also in response to 3D stiffness macropatterns, dependent on the stimulus timing and temporally followed by (slower) ECM co-alignment. In conclusion, the developed model systems allow improved fundamental understanding of the underlying mechanobiological factors that steer cell and ECM orientation, such as stiffness guidance and boundary constraints.

A multiscale computational model of arterial growth and remodeling including Notch signaling.

Blood vessels grow and remodel in response to mechanical stimuli. Many computational models capture this process phenomenologically, by assuming stress homeostasis, but this approach cannot unravel the underlying cellular mechanisms. Mechano-sensitive Notch signaling is well-known to be key in vascular development and homeostasis. Here, we present a multiscale framework coupling a constrained mixture model, capturing the mechanics and turnover of arterial constituents, to a cell-cell signaling model, describing Notch signaling dynamics among vascular smooth muscle cells (SMCs) as influenced by mechanical stimuli. Tissue turnover was regulated by both Notch activity, informed by in vitro data, and a phenomenological contribution, accounting for mechanisms other than Notch. This novel framework predicted changes in wall thickness and arterial composition in response to hypertension similar to previous in vivo data. The simulations suggested that Notch contributes to arterial growth in hypertension mainly by promoting SMC proliferation, while other mechanisms are needed to fully capture remodeling. The results also indicated that interventions to Notch, such as external Jagged ligands, can alter both the geometry and composition of hypertensive vessels, especially in the short term. Overall, our model enables a deeper analysis of the role of Notch and Notch interventions in arterial growth and remodeling and could be adopted to investigate therapeutic strategies and optimize vascular regeneration protocols.

Bmp9 regulates Notch signaling and the temporal dynamics of angiogenesis via Lunatic Fringe.

In brief: The mechanisms regulating the signaling pathways involved in angiogenesis are not fully known. Ristori et al. show that Lunatic Fringe (LFng) mediates the crosstalk between Bone Morphogenic Protein 9 (Bmp9) and Notch signaling, thereby regulating the endothelial cell behavior and temporal dynamics of their identity during sprouting angiogenesis. Highlights: Bmp9 upregulates the expression of LFng in endothelial cells.LFng regulates the temporal dynamics of tip/stalk selection and rearrangement.LFng indicated to play a role in hereditary hemorrhagic telangiectasia.Bmp9 and LFng mediate the endothelial cell-pericyte crosstalk.Bone Morphogenic Protein 9 (Bmp9), whose signaling through Activin receptor-like kinase 1 (Alk1) is involved in several diseases, has been shown to independently activate Notch target genes in an additive fashion with canonical Notch signaling. Here, by integrating predictive computational modeling validated with experiments, we uncover that Bmp9 upregulates Lunatic Fringe (LFng) in endothelial cells (ECs), and thereby also regulates Notch activity in an inter-dependent, multiplicative fashion. Specifically, the Bmp9-upregulated LFng enhances Notch receptor activity creating a much stronger effect when Dll4 ligands are also present. During sprouting, this LFng regulation alters vessel branching by modulating the timing of EC phenotype selection and rearrangement. Our results further indicate that LFng can play a role in Bmp9-related diseases and in pericyte-driven vessel stabilization, since we find LFng contributes to Jag1 upregulation in Bmp9-stimulated ECs; thus, Bmp9-upregulated LFng results in not only enhanced EC Dll4-Notch1 activation, but also Jag1-Notch3 activation in pericytes.

Mechano-regulated cell-cell signaling in the context of cardiovascular tissue engineering.

Cardiovascular tissue engineering (CVTE) aims to create living tissues, with the ability to grow and remodel, as replacements for diseased blood vessels and heart valves. Despite promising results, the (long-term) functionality of these engineered tissues still needs improvement to reach broad clinical application. The functionality of native tissues is ensured by their specific mechanical properties directly arising from tissue organization. We therefore hypothesize that establishing a native-like tissue organization is vital to overcome the limitations of current CVTE approaches. To achieve this aim, a better understanding of the growth and remodeling (G&R) mechanisms of cardiovascular tissues is necessary. Cells are the main mediators of tissue G&R, and their behavior is strongly influenced by both mechanical stimuli and cell-cell signaling. An increasing number of signaling pathways has also been identified as mechanosensitive. As such, they may have a key underlying role in regulating the G&R of tissues in response to mechanical stimuli. A more detailed understanding of mechano-regulated cell-cell signaling may thus be crucial to advance CVTE, as it could inspire new methods to control tissue G&R and improve the organization and functionality of engineered tissues, thereby accelerating clinical translation. In this review, we discuss the organization and biomechanics of native cardiovascular tissues; recent CVTE studies emphasizing the obtained engineered tissue organization; and the interplay between mechanical stimuli, cell behavior, and cell-cell signaling. In addition, we review past contributions of computational models in understanding and predicting mechano-regulated tissue G&R and cell-cell signaling to highlight their potential role in future CVTE strategies.

Notch signaling regulates strain-mediated phenotypic switching of vascular smooth muscle cells.

Mechanical stimuli experienced by vascular smooth muscle cells (VSMCs) and mechanosensitive Notch signaling are important regulators of vascular growth and remodeling. However, the interplay between mechanical cues and Notch signaling, and its contribution to regulate the VSMC phenotype are still unclear. Here, we investigated the role of Notch signaling in regulating strain-mediated changes in VSMC phenotype. Synthetic and contractile VSMCs were cyclically stretched for 48 h to determine the temporal changes in phenotypic features. Different magnitudes of strain were applied to investigate its effect on Notch mechanosensitivity and the phenotypic regulation of VSMCs. In addition, Notch signaling was inhibited via DAPT treatment and activated with immobilized Jagged1 ligands to understand the role of Notch on strain-mediated phenotypic changes of VSMCs. Our data demonstrate that cyclic strain induces a decrease in Notch signaling along with a loss of VSMC contractile features. Accordingly, the activation of Notch signaling during cyclic stretching partially rescued the contractile features of VSMCs. These findings demonstrate that Notch signaling has an important role in regulating strain-mediated phenotypic switching of VSMCs.

Computational analysis of the role of mechanosensitive Notch signaling in arterial adaptation to hypertension.

Arteries grow and remodel in response to mechanical stimuli. Hypertension, for example, results in arterial wall thickening. Cell-cell Notch signaling between vascular smooth muscle cells (VSMCs) is known to be involved in this process, but the underlying mechanisms are still unclear. Here, we investigated whether Notch mechanosensitivity to strain may regulate arterial thickening in hypertension. We developed a multiscale computational framework by coupling a finite element model of arterial mechanics, including residual stress, to an agent-based model of mechanosensitive Notch signaling, to predict VSMC phenotypes as an indicator of growth and remodeling. Our simulations revealed that the sensitivity of Notch to strain at mean blood pressure may be a key mediator of arterial thickening in hypertensive arteries. Further simulations showed that loss of residual stress can have synergistic effects with hypertension, and that changes in the expression of Notch receptors, but not Jagged ligands, may be used to control arterial growth and remodeling and to intensify or counteract hypertensive thickening. Overall, we identify Notch mechanosensitivity as a potential mediator of vascular adaptation, and we present a computational framework that can facilitate the testing of new therapeutic and regenerative strategies.

Engineered patterns of Notch ligands Jag1 and Dll4 elicit differential spatial control of endothelial sprouting.

Spatial regulation of angiogenesis is important for the generation of functional engineered vasculature in regenerative medicine. The Notch ligands Jag1 and Dll4 show distinct expression patterns in endothelial cells and, respectively, promote and inhibit endothelial sprouting. Therefore, patterns of Notch ligands may be utilized to spatially control sprouting, but their potential and the underlying mechanisms of action are unclear. Here, we coupled in vitro and in silico models to analyze the ability of micropatterned Jag1 and Dll4 ligands to spatially control endothelial sprouting. Dll4 patterns, but not Jag1 patterns, elicited spatial control. Computational simulations of the underlying signaling dynamics suggest that different timing of Notch activation by Jag1 and Dll4 underlie their distinct ability to spatially control sprouting. Hence, Dll4 patterns efficiently direct the sprouts, whereas longer exposure to Jag1 patterns is required to achieve spatial control. These insights in sprouting regulation offer therapeutic handles for spatial regulation of angiogenesis.

Ex Vivo Models to Decipher the Molecular Mechanisms of Genetic Notch Cardiovascular Disorders.

Notch is an evolutionary, conserved, cell-cell signaling pathway that is central to several biological processes, from tissue morphogenesis to homeostasis. It is therefore not surprising that several genetic mutations of Notch components cause inherited human diseases, especially cardiovascular disorders. Despite numerous efforts, current in vivo models are still insufficient to unravel the underlying mechanisms of these pathologies, hindering the development of utmost needed medical therapies. In this perspective review, we discuss the limitations of current murine models and outline how the combination of microphysiological systems (MPSs) and targeted computational models can lead to breakthroughs in this field. In particular, while MPSs enable the experimentation on human cells in controlled and physiological environments, in silico models can provide a versatile tool to translate the in vitro findings to the more complex in vivo setting. As a showcase example, we focus on Notch-related cardiovascular diseases, such as Alagille syndrome, Adams-Oliver syndrome, and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Impact statement In this review, a comprehensive overview of the limitations of current in vivo models of genetic Notch cardiovascular diseases is provided, followed by a discussion over the potential of microphysiological systems and computational models in overcoming these limitations and in potentiating drug testing and modeling of these pathologies.

Computational modeling for cardiovascular tissue engineering: the importance of including cell behavior in growth and remodeling algorithms.

Understanding cardiovascular growth and remodeling (G&R) is fundamental for designing robust cardiovascular tissue engineering strategies, which enable synthetic or biological scaffolds to transform into healthy living tissues after implantation. Computational modeling, particularly when integrated with experimental research, is key for advancing our understanding, predicting the in vivo evolution of engineered tissues, and efficiently optimizing scaffold designs. As cells are ultimately the drivers of G&R and known to change their behavior in response to mechanical cues, increasing efforts are currently undertaken to capture (mechano-mediated) cell behavior in computational models. In this selective review, we highlight some recent examples that are relevant in the context of cardiovascular tissue engineering and discuss the current and future biological and computational challenges for modeling cell-mediated G&R.

Lateral induction limits the impact of cell connectivity on Notch signaling in arterial walls.

It is well known that arteries grow and remodel in response to mechanical stimuli. Vascular smooth muscle cells are the main mediators of this process, as they can switch phenotype from contractile to synthetic, and vice-versa, based on the surrounding bio-chemo-mechanical stimuli. A correct regulation of this phenotypic switch is fundamental to obtain and maintain arterial homeostasis. Notch, a mechanosensitive signaling pathway, is one of the main regulators of the vascular smooth muscle cell phenotype. Therefore, understanding Notch dynamics is key to elucidate arterial growth, remodeling, and mechanobiology. We have recently developed a one-dimensional agent-based model to investigate Notch signaling in arteries. However, due to its one-dimensional formulation, the model cannot be adopted to study complex nonsymmetrical geometries and, importantly, it cannot capture the realistic "cell connectivity" in arteries, here defined as the number of cell neighbors. Notch functions via direct cell-cell contact; thus, the number of cell neighbors could be an essential feature of Notch dynamics. Here, we extended the agent-based model to a two-dimensional formulation, to investigate the effects of cell connectivity on Notch dynamics and cell phenotypes in arteries. The computational results, supported by a sensitivity analysis, indicate that cell connectivity has marginal effects when Notch dynamics is dominated by the process of lateral induction, which induces all cells to have a uniform phenotype. When lateral induction is weaker, cells exhibit a nonuniform phenotype distribution and the percentage of synthetic cells within an artery depends on the number of neighbors.

Pressure-induced collagen degradation in arterial tissue as a potential mechanism for degenerative arterial disease progression.

Collagen fibre degradation is a strain-dependent process, whereby the magnitude of experienced strain dictates the rate of enzymatic cleavage. Studies have identified conflicting findings as to whether strain inhibits or enhances collagen degradation, which may be explained by the tissue type and tissue scale investigated, as well as the strain range considered. The aim of this study is to identify, for the first time, the strain-dependent degradation response of intact arterial vessels experiencing physiological pressures and apply these findings to a computational model to better understand degenerative arterial diseases, such as aneurysms. To achieve this, a series of quasi-static pressure inflation experiments were carried out on intact arteries in the presence of purified bacterial collagenase at physiologically relevant pressures to investigate collagen matrix degradation in the vascular wall. A complementary computational model was developed to explore the complex role of pressure, non-collagenous matrix contribution, and collagen fibre crimp in the ultimate degradation response of the vessel. Pressure induced inflation-degradation results identified an increased rate of vessel expansion and reduced time to failure with increasing pressure in the vessels. Interestingly, our computational model was able to capture this same response, including the elevated rates of degradation which occur at low pressures. These findings highlight the critical role of strain in collagen degradation, particularly in cases of arterial disease, such as aneurysm formation, whereby structural integrity may be compromised.

Cellular Contact Guidance Emerges from Gap Avoidance.

In the presence of anisotropic biochemical or topographical patterns, cells tend to align in the direction of these cues-a widely reported phenomenon known as "contact guidance." To investigate the origins of contact guidance, here, we created substrates micropatterned with parallel lines of fibronectin with dimensions spanning multiple orders of magnitude. Quantitative morphometric analysis of our experimental data reveals two regimes of contact guidance governed by the length scale of the cues that cannot be explained by enforced alignment of focal adhesions. Adopting computational simulations of cell remodeling on inhomogeneous substrates based on a statistical mechanics framework for living cells, we show that contact guidance emerges from anisotropic cell shape fluctuation and "gap avoidance," i.e., the energetic penalty of cell adhesions on non-adhesive gaps. Our findings therefore point to general biophysical mechanisms underlying cellular contact guidance, without the necessity of invoking specific molecular pathways.

Vimentin regulates Notch signaling strength and arterial remodeling in response to hemodynamic stress.

The intermediate filament (IF) cytoskeleton has been proposed to regulate morphogenic processes by integrating the cell fate signaling machinery with mechanical cues. Signaling between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) through the Notch pathway regulates arterial remodeling in response to changes in blood flow. Here we show that the IF-protein vimentin regulates Notch signaling strength and arterial remodeling in response to hemodynamic forces. Vimentin is important for Notch transactivation by ECs and vimentin knockout mice (VimKO) display disrupted VSMC differentiation and adverse remodeling in aortic explants and in vivo. Shear stress increases Jagged1 levels and Notch activation in a vimentin-dependent manner. Shear stress induces phosphorylation of vimentin at serine 38 and phosphorylated vimentin interacts with Jagged1 and increases Notch activation potential. Reduced Jagged1-Notch transactivation strength disrupts lateral signal induction through the arterial wall leading to adverse remodeling. Taken together we demonstrate that vimentin forms a central part of a mechanochemical transduction pathway that regulates multilayer communication and structural homeostasis of the arterial wall.

A computational analysis of cell-mediated compaction and collagen remodeling in tissue-engineered heart valves.

One of the most critical problems in heart valve tissue engineering is the progressive development of valvular insufficiency due to leaflet retraction. Understanding the underlying mechanisms of this process is crucial for developing tissue-engineered heart valves (TEHVs) that maintain their functionality in the long term. In the present study, we adopted a computational approach to predict the remodeling process in TEHVs subjected to dynamic pulmonary and aortic pressure conditions, and to assess the risk of valvular insufficiency. In addition, we investigated the importance of the intrinsic cell contractility on the final outcome of the remodeling process. For valves implanted in the aortic position, the model predictions suggest that valvular insufficiency is not likely to occur as the blood pressure is high enough to prevent the development of leaflet retraction. In addition, the collagen network was always predicted to remodel towards a circumferentially aligned network, which is corresponding to the native situation. In contrast, for valves implanted in the pulmonary position, our model predicted that there is a high risk for the development of valvular insufficiency, unless the cell contractility is very low. Conversely, the development of a circumferential collagen network was only predicted at these pressure conditions when cell contractility was high. Overall, these results, therefore, suggest that tissue remodeling at aortic pressure conditions is much more stable and favorable compared to tissue remodeling at pulmonary pressure conditions.