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.

Mechanosensitivity of Jagged-Notch signaling can induce a switch-type behavior in vascular homeostasis.

Hemodynamic forces and Notch signaling are both known as key regulators of arterial remodeling and homeostasis. However, how these two factors integrate in vascular morphogenesis and homeostasis is unclear. Here, we combined experiments and modeling to evaluate the impact of the integration of mechanics and Notch signaling on vascular homeostasis. Vascular smooth muscle cells (VSMCs) were cyclically stretched on flexible membranes, as quantified via video tracking, demonstrating that the expression of Jagged1, Notch3, and target genes was down-regulated with strain. The data were incorporated in a computational framework of Notch signaling in the vascular wall, where the mechanical load was defined by the vascular geometry and blood pressure. Upon increasing wall thickness, the model predicted a switch-type behavior of the Notch signaling state with a steep transition of synthetic toward contractile VSMCs at a certain transition thickness. These thicknesses varied per investigated arterial location and were in good agreement with human anatomical data, thereby suggesting that the Notch response to hemodynamics plays an important role in the establishment of vascular homeostasis.

GFAP alternative splicing regulates glioma cell-ECM interaction in a DUSP4-dependent manner.

Gliomas are the most common primary brain tumors. Their highly invasive character and the heterogeneity of active oncogenic pathways within single tumors complicate the development of curative therapies and cause poor patient prognosis. Glioma cells express the intermediate filament protein glial fibrillary acidic protein (GFAP), and the level of its alternative splice variant GFAP-δ, relative to its canonical splice variant GFAP-α, is higher in grade IV compared with lower-grade and lower malignant glioma. In this study we show that a high GFAP-δ/α ratio induces the expression of the dual-specificity phosphatase 4 (DUSP4) in focal adhesions. By focusing on pathways up- and downstream of DUSP4 that are involved in the cell-extracellular matrix interaction, we show that a high GFAP-δ/α ratio equips glioma cells to better invade the brain. This study supports the hypothesis that glioma cells with a high GFAP-δ/α ratio are highly invasive and more malignant cells, thus making GFAP alternative splicing a potential therapeutic target.-Van Bodegraven, E. J., van Asperen, J. V., Sluijs, J. A., van Deursen, C. B. J., van Strien, M. E., Stassen, O. M. J. A., Robe, P. A. J., Hol, E. M. GFAP alternative splicing regulates glioma cell-ECM interaction in a DUSP4-dependent manner.

The Mechanical Contribution of Vimentin to Cellular Stress Generation.

Contractile stress generation by adherent cells is largely determined by the interplay of forces within their cytoskeleton. It is known that actin stress fibers, connected to focal adhesions, provide contractile stress generation, while microtubules and intermediate filaments provide cells compressive stiffness. Recent studies have shown the importance of the interplay between the stress fibers and the intermediate filament vimentin. Therefore, the effect of the interplay between the stress fibers and vimentin on stress generation was quantified in this study. We hypothesized that net stress generation comprises the stress fiber contraction combined with the vimentin resistance. We expected an increased net stress in vimentin knockout (VimKO) mouse embryonic fibroblasts (MEFs) compared to their wild-type (vimentin wild-type (VimWT)) counterparts, due to the decreased resistance against stress fiber contractility. To test this, the net stress generation by VimKO and VimWT MEFs was determined using the thin film method combined with sample-specific finite element modeling. Additionally, focal adhesion and stress fiber organization were examined via immunofluorescent staining. Net stress generation of VimKO MEFs was three-fold higher compared to VimWT MEFs. No differences in focal adhesion size or stress fiber organization and orientation were found between the two cell types. This suggests that the increased net stress generation in VimKO MEFs was caused by the absence of the resistance that vimentin provides against stress fiber contraction. Taken together, these data suggest that vimentin resists the stress fiber contractility, as hypothesized, thus indicating the importance of vimentin in regulating cellular stress generation by adherent cells.

GFAP isoforms control intermediate filament network dynamics, cell morphology, and focal adhesions.

Glial fibrillary acidic protein (GFAP) is the characteristic intermediate filament (IF) protein in astrocytes. Expression of its main isoforms, GFAPα and GFAPδ, varies in astrocytes and astrocytoma implying a potential regulatory role in astrocyte physiology and pathology. An IF-network is a dynamic structure and has been functionally linked to cell motility, proliferation, and morphology. There is a constant exchange of IF-proteins with the network. To study differences in the dynamic properties of GFAPα and GFAPδ, we performed fluorescence recovery after photobleaching experiments on astrocytoma cells with fluorescently tagged GFAPs. Here, we show for the first time that the exchange of GFP-GFAPδ was significantly slower than the exchange of GFP-GFAPα with the IF-network. Furthermore, a collapsed IF-network, induced by GFAPδ expression, led to a further decrease in fluorescence recovery of both GFP-GFAPα and GFP-GFAPδ. This altered IF-network also changed cell morphology and the focal adhesion size, but did not alter cell migration or proliferation. Our study provides further insight into the modulation of the dynamic properties and functional consequences of the IF-network composition.

Silencing GFAP isoforms in astrocytoma cells disturbs laminin-dependent motility and cell adhesion.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed in astrocytes and neural stem cells. The GFAP gene is alternatively spliced, and expression of GFAP is highly regulated during development, on brain damage, and in neurodegenerative diseases. GFAPα is the canonical splice variant and is expressed in all GFAP-positive cells. In the human brain, the alternatively spliced transcript GFAPδ marks specialized astrocyte populations, such as subpial astrocytes and the neurogenic astrocytes in the human subventricular zone. We here show that shifting the GFAP isoform ratio in favor of GFAPδ in astrocytoma cells, by selectively silencing the canonical isoform GFAPα with short hairpin RNAs, induced a change in integrins, a decrease in plectin, and an increase in expression of the extracellular matrix component laminin. Together, this did not affect cell proliferation but resulted in a significantly decreased motility of astrocytoma cells. In contrast, a down-regulation of all GFAP isoforms led to less cell spreading, increased integrin expression, and a >100-fold difference in the adhesion of astrocytoma cells to laminin. In summary, isoform-specific silencing of GFAP revealed distinct roles of a specialized GFAP network in regulating the interaction of astrocytoma cells with the extracellular matrix through laminin.-Moeton, M., Kanski, R., Stassen, O. M. J. A., Sluijs, J. A., Geerts, D., van Tijn, P., Wiche, G., van Strien, M. E., Hol, E. M. Silencing GFAP isoforms in astrocytoma cells disturbs laminin dependent motility and cell adhesion.

Engineering a scalable and orthogonal platform for synthetic communication in mammalian cells.

The rational design and implementation of synthetic mammalian communication systems can unravel fundamental design principles of cell communication circuits and offer a framework for engineering of designer cell consortia with potential applications in cell therapeutics. Here, we develop the foundations of an orthogonal, and scalable mammalian synthetic communication platform that exploits the programmability of synthetic receptors and selective affinity and tunability of diffusing coiled-coil peptides. Leveraging the ability of coiled-coils to exclusively bind to a cognate receptor, we demonstrate orthogonal receptor activation and Boolean logic operations at the receptor level. We show intercellular communication based on synthetic receptors and secreted multidomain coiled-coils and demonstrate a three-cell population system that can perform AND gate logic. Finally, we show CC-GEMS receptor-dependent therapeutic protein expression. Our work provides a modular and scalable framework for the engineering of complex cell consortia, with the potential to expand the aptitude of cell therapeutics and diagnostics.

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.

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.

A Supramolecular Platform for the Introduction of Fc-Fusion Bioactive Proteins on Biomaterial Surfaces.

Bioorthogonal chemistry is an excellent method for functionalization of biomaterials with bioactive molecules, as it allows for decoupling of material processing and bioactivation. Here, we report on a modular system created by means of tetrazine/trans-cyclooctene (Tz/TCO) click chemistry undergoing an inverse electron demand Diels-Alder cycloaddition. A reactive supramolecular surface based on ureido-pyrimidinones (UPy) is generated via a UPy-Tz additive, in order to introduce a versatile TCO-protein G conjugate for immobilization of Fc-fusion proteins. As a model bioactive protein, we introduced Fc-Jagged1, a Notch ligand, to induce Notch signaling activity on the material. Interestingly, HEK293 FLN1 cells expressing the Notch1 receptor were repelled by films modified with TCO-protein G but adhered and spread on functionalized electrospun meshes. This indicates that the material processing method influences the biocompatibility of the postmodification. Notch signaling activity was upregulated 5.6-fold with respect to inactive controls on electrospun materials modified with TCO-protein G/Fc-Jagged1. Furthermore, downstream effects of Notch signaling were detected on the gene level in vascular smooth muscle cells expressing the Notch3 receptor. Taken together, our results demonstrate the successful use of a modular supramolecular system for the postprocessing modification of solid materials with functional proteins.

Influence of the Assembly State on the Functionality of a Supramolecular Jagged1-Mimicking Peptide Additive.

Expanding the bioactivation toolbox of supramolecular materials is of utmost relevance for their broad applicability in regenerative medicines. This study explores the functionality of a peptide mimic of the Notch ligand Jagged1 in a supramolecular system that is based on hydrogen bonding ureido-pyrimidinone (UPy) units. The functionality of the peptide is studied when formulated as an additive in a supramolecular solid material and as a self-assembled system in solution. UPy conjugation of the DSLJAG1 peptide sequence allows for the supramolecular functionalization of UPy-modified polycaprolactone, an elastomeric material, with UPy-DSLJAG1. Surface presentation of the UPy-DSLJAG1 peptide was confirmed by atomic force microscopy and X-ray photoelectron spectroscopy analyses, but no enhancement of Notch activity was detected in cells presenting Notch1 and Notch3 receptors. Nevertheless, a significant increase in Notch-signaling activity was observed when DSLJAG1 peptides were administered in the soluble form, indicating that the activity of DSLJAG1 is preserved after UPy functionalization but not after immobilization on a supramolecular solid material. Interestingly, an enhanced activity in solution of the UPy conjugate was detected compared with the unconjugated DSLJAG1 peptide, suggesting that the self-assembly of supramolecular aggregates in solution ameliorates the functionality of the molecules in a biological context.

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 biomimetic microfluidic model to study signalling between endothelial and vascular smooth muscle cells under hemodynamic conditions.

Cell signalling and mechanics influence vascular pathophysiology and there is an increasing demand for in vitro model systems that enable examination of signalling between vascular cells under hemodynamic conditions. Current 3D vessel wall constructs do not recapitulate the mechanical conditions of the native tissue nor do they allow examination of cell-cell interactions under relevant hemodynamic conditions. Here, we describe a 3D microfluidic chip model of arterial endothelial and smooth muscle cells where cellular organization, composition and interactions, as well as the mechanical environment of the arterial wall are mimicked. The hemodynamic EC-VSMC-signalling-on-a-chip consists of two parallel polydimethylsiloxane (PDMS) cell culture channels, separated by a flexible, porous PDMS membrane, mimicking the porosity of the internal elastic lamina. The hemodynamic EC-VSMC-signalling-on-a-chip allows co-culturing of human aortic endothelial cells (ECs) and human aortic vascular smooth muscle cells (VSMCs), separated by a porous membrane, which enables EC-VSMC interaction and signalling, crucial for the development and homeostasis of the vessel wall. The device allows real time cell imaging and control of hemodynamic conditions. The culture channels are surrounded on either side by vacuum channels to induce cyclic strain by applying cyclic suction, resulting in mechanical stretching and relaxation of the membrane in the cell culture channels. The blood flow is mimicked by creating a flow of medium at the EC side. Vascular cells remain viable during prolonged culturing, exhibit physiological morphology and organization and make cell-cell contact. During dynamic culturing of the device with a shear stress of 1-1.5 Pa and strain of 5-8%, VSMCs align perpendicular to the given strain in the direction of the flow and EC adopt a cobblestone morphology. To our knowledge, this is the first report on the development of a microfluidic device, which enables a co-culture of interacting ECs and VSMCs under hemodynamic conditions and presents a novel approach to systematically study the biological and mechanical components of the intimal-medial vascular unit.

GFAPδ/GFAPα ratio directs astrocytoma gene expression towards a more malignant profile.

Astrocytomas are the most common malignant brain tumours and are to date incurable. It is unclear how astrocytomas progress into higher malignant grades. The intermediate filament cytoskeleton is emerging as an important regulator of malignancy in several tumours. The majority of the astrocytomas express the intermediate filament protein Glial Fibrillary Acidic Protein (GFAP). Several GFAP splice variants have been identified and the main variants expressed in human astrocytoma are the GFAPα and GFAPδ isoforms. Here we show a significant downregulation of GFAPα in grade IV astrocytoma compared to grade II and III, resulting in an increased GFAPδ/α ratio. Mimicking this increase in GFAPδ/α ratio in astrocytoma cell lines and comparing the subsequent transcriptomic changes with the changes in the patient tumours, we have identified a set of GFAPδ/α ratio-regulated high-malignant and low-malignant genes. These genes are involved in cell proliferation and protein phosphorylation, and their expression correlated with patient survival. We additionally show that changing the ratio of GFAPδ/α, by targeting GFAP expression, affected expression of high-malignant genes. Our data imply that regulating GFAP expression and splicing are novel therapeutic targets that need to be considered as a treatment for astrocytoma.

Enhanced transduction of CAR-negative cells by protein IX-gene deleted adenovirus 5 vectors.

In human adenoviruses (HAdV), 240 copies of the 14.3-kDa minor capsid protein IX stabilize the capsid. Three N-terminal domains of protein IX form triskelions between hexon capsomers. The C-terminal domains of four protein IX monomers associate near the facet periphery. The precise biological role of protein IX remains enigmatic. Here we show that deletion of the protein IX gene from a HAdV-5 vector enhanced the reporter gene delivery 5 to 25-fold, specifically to Coxsackie and Adenovirus Receptor (CAR)-negative cell lines. Deletion of the protein IX gene also resulted in enhanced activation of peripheral blood mononuclear cells. The mechanism for the enhanced transduction is obscure. No differences in fiber loading, integrin-dependency of transduction, or factor-X binding could be established between protein IX-containing and protein IX-deficient particles. Our data suggest that protein IX can affect the cell tropism of HAdV-5, and may function to dampen the innate immune responses against HAdV particles.