Biology subjects

Bouten, C. V. C.

Publications and source records attributed to Bouten, C. V. C..

6 recordsLinked to original sources

bioRxiv2025

Mechanosensitive interactions between Jag1 and Myo1c control Jag1 trafficking in endothelial cells

Morphogenesis of the cardiovascular system is responsive to hemodynamic cues. In endothelial cells the organization of morphogenic signaling proteins can be regulated by membrane presentation and internalization of proteins. How these signaling proteins are regulated by hemodynamics is unclear. One of the signaling proteins that is regulated by hemodynamics is Jagged1, a ligand in the Notch pathway. Here we set out to identify factors that differentially interact with Jagged1 in response to shear stress exposure, by combining an orbital shaker as a shear stress platform with endothelial cells expressing Jagged1 coupled to an APEX2-tag for proximity labeling. Myo1c was identified and confirmed through coimmunoprecipitation as a Jag1 interacting factor under static conditions, with reduced interaction after exposure to shear in endothelial cells. We showed that Jagged1 polarized downstream of shear followed by nucleograde transport of Jagged1. Myo1c knockout inhibited shear-induced Jagged1 polarization and consequent nucleograde transport. Further, Myo1c knockdown reduced membrane levels of Jagged1 under static conditions, but not under shear conditions. Together, our data reveal a role for Myo1c in the hemodynamic control of Jagged1 localization in endothelial cells.

cell biology↗

bioRxiv2024

Jagged1 is a Notch-independent mechanotransducer in endothelial cells

The Notch signaling pathway plays a crucial role in regulating endothelial biology. Notch signaling is sensitive to hemodynamic forces and governs mechanically-driven cardiovascular development, physiology, and remodeling. However, the mechanisms by which mechanical forces integrate with the Notch pathway remain largely unknown. Here, we uncover a non-canonical role for the Notch ligand Jagged1 in regulating the activity of mechanosensitive kinases in endothelial cells. We show that stress induces expression and relocalization of Jagged1 to cell junctions downstream of flow. Jagged1 expression under stress demonstrates magnitude dependence and peaks at 0.8-1Pa without impacting Jagged1s Notch-activation potential. On the contrary Jagged1 regulates the activity of mechanosensitive kinases. Deletion of Jagged1 reduces the activity of VEGFR2 and ERK in vitro and diminished ERK activity in zebrafish embryos without affecting canonical Notch signaling. Furthermore, the direct physical stimulation of Jagged1 using antibody-conjugated beads triggers the activation of VEGFR2 and ERK, mediated by Jagged1-induces Src activation. Taken together, we demonstrate a novel non-canonical role for Jagged1 as a regulator of the activity of pathways involved in endothelial mechanotransduction.

cell biology↗

bioRxiv2022

Transiently heritable fates and quorum sensing drive early IFN-I response dynamics

Type I Interferon (IFN-I)-mediated antiviral responses are central to host defense against viral infections. Crucial is the tight and well-orchestrated control of cellular decision-making leading to the production of IFN-Is. Innovative single-cell approaches revealed that the initiation of IFN-I production is only limited to a fraction of 1-3% of the total population, both found in vitro and in vivo, which were thought to be stochastically regulated. To challenge this dogma, we addressed the influence of various host-intrinsic factors, both stochastic and epigenetic, on dictating early IFN-I responses. Hypomethylating drugs increased the percentage of responding cells. Next, with the classical Luria-Delbruck fluctuation test, we provided evidence that the fate of becoming a responding cell is transiently heritable. Finally, while studying varying cell-densities, we substantiated an important role for quorum sensing, which was verified by mathematical modeling. Together, this systems immunology approach opens up new avenues to progress the fundamental understanding of cellular decision-making during early IFN-I responses, which can be translated to other (immune) signaling systems, and ultimately will improve IFN-I based immune therapies.

immunology↗

bioRxiv2021

Tissue-engineered collagenous fibrous cap models to systematically elucidate atherosclerotic plaque rupture.

A significant amount of vascular thrombotic events is associated with rupture of the fibrous cap that overlie atherosclerotic plaques. Cap rupture is however difficult to predict due to the heterogenous composition of the plaque, unknown material properties, and the stochastic nature of the event. Here, we aim to create tissue engineered human fibrous cap models with a variable but controllable collagen composition, suitable for mechanical testing, to scrutinize the reciprocal relationships between composition and mechanical properties. Myofibroblasts were cultured in 1 x 1.5 cm-sized fibrin-based constrained gels for 21 days according to established (dynamic) culture protocols (i.e. static, intermittent or continuous loading) to vary collagen composition (e.g. amount, type and organization). At day 7, a soft 2 mm O fibrin inclusion was introduced in the centre of each tissue to mimic the soft lipid core, simulating the heterogeneity of a plaque. Results demonstrate reproducible collagenous tissues, that mimic the bulk mechanical properties of human caps and vary in collagen composition due to the presence of an successfully integrated soft inclusion and the culture protocol applied. The models can be deployed to assess tissue mechanics, evolution and failure of fibrous caps or complex heterogeneous tissues in general.

bioengineering↗

bioRxiv2021

Inflammatory and regenerative processes in bioresorbable synthetic pulmonary valves up to 2 years in sheep: Spatiotemporal insights augmented by Raman microspectroscopy

In situ heart valve tissue engineering is an emerging approach in which resorbable, off-the-shelf available scaffolds are used to induce endogenous heart valve restoration. Such scaffolds are designed to recruit endogenous cells in vivo, which subsequently resorb polymer and produce and remodel new valvular tissue in situ. Recently, preclinical studies using electrospun supramolecular elastomeric valvular grafts have shown that this approach enables in situ regeneration of pulmonary valves with long-term functionality in vivo. However, the evolution and mechanisms of inflammation, polymer absorption and tissue regeneration are largely unknown, and adverse valve remodeling and intra- and inter-valvular variability have been reported. Therefore, the goal of the present study was to gain a mechanistic understanding of the in vivo regenerative processes by combining routine histology and immunohistochemistry, using a comprehensive sheep-specific antibody panel, with Raman microspectroscopy for the spatiotemporal analysis of in situ tissue-engineered pulmonary valves with follow-up to 24 months from a previous preclinical study in sheep. The analyses revealed a strong spatial heterogeneity in the influx of inflammatory cells, graft resorption, and foreign body giant cells. Collagen maturation occurred predominantly between 6 and 12 months after implantation, which was accompanied by a progressive switch to a more quiescent phenotype of infiltrating cells with properties of valvular interstitial cells. Variability among specimens in the extent of tissue remodeling was observed for follow-up times after 6 months. Taken together, these findings advance the understanding of key events and mechanisms in material-driven in situ heart valve tissue engineering.

bioengineering↗

bioRxiv2019

Human in vitro model of material-driven vascular regeneration reveals how cyclic stretch and shear stress differentially modulate inflammation and tissue formation

1Resorbable synthetic scaffolds designed to regenerate living tissues and organs inside the body emerge as a clinically attractive technology to replace diseased blood vessels. However, mismatches between scaffold design and in vivo hemodynamic loading (i.e., cyclic stretch and shear stress) can result in aberrant inflammation and adverse tissue remodeling, leading to premature graft failure. Yet, the underlying mechanisms remain elusive. Here, a human in vitro model is presented that mimics the transient local inflammatory and biomechanical environments that drive scaffold-guided tissue regeneration. The model is based on the co-culture of human (myo)fibroblasts and macrophages in a bioreactor platform that decouples cyclic stretch and shear stress. Using a resorbable supramolecular elastomer as the scaffold material, it is revealed that cyclic stretch initially reduces pro-inflammatory cytokine secretion and, especially when combined with shear stress, stimulates IL-10 secretion. Moreover, cyclic stretch stimulates downstream (myo)fibroblast proliferation and neotissue formation. In turn, shear stress attenuates cyclic-stretch-induced tissue growth by enhancing MMP-1/TIMP-1-mediated collagen remodeling, and synergistically alters (myo)fibroblast phenotype when combined with cyclic stretch. The findings suggest that shear stress acts as a stabilizing factor in cyclic stretch-induced tissue formation and highlight the distinct roles of hemodynamic loads in the design of resorbable vascular grafts.

bioengineering↗