Hypertensive pressure mechanosensing alone triggers lipid droplet accumulation and transdifferentiation of vascular smooth muscle cells to foam cells

Arterial Vascular smooth muscle cells (VSMCs) play a central role in the onset and progression of atherosclerosis. Upon exposure to pathological stimuli, they can take on alternative phenotypes that, among others, have been described as macrophage like, or foam cells. VSMC foam cells make up >50% of all arterial foam cells and have been suggested to retain an even higher proportion of the cell stored lipid droplets, further leading to apoptosis, secondary necrosis, and an inflammatory response. However, the mechanism of VSMC foam cell formation is still unclear. Here, it is identified that mechanical stimulation through hypertensive pressure alone is sufficient for the phenotypic switch. Hyperspectral stimulated Raman scattering imaging demonstrates rapid lipid droplet formation and changes to lipid metabolism and changes are confirmed in ABCA1, KLF4, LDLR, and CD68 expression, cell proliferation, and migration. Further, a mechanosignaling route is identified involving Piezo1, phospholipid, and arachidonic acid signaling, as well as epigenetic regulation, whereby CUT&Tag epigenomic analysis confirms changes in the cells (lipid) metabolism and atherosclerotic pathways. Overall, the results show for the first time that VSMC foam cell formation can be triggered by mechanical stimulation alone, suggesting modulation of mechanosignaling can be harnessed as potential therapeutic strategy

Regulation of cardiomyocyte adhesion and mechanosignalling through distinct nanoscale behaviour of integrin ligands mimicking healthy or fibrotic ECM

No abstract available

Mécanotransduction des cellules souches de glioblastome dans un nouveau modèle de culture tridimensionnel : implication de la MGAT5 dans la perception de l'environnement mécanique

Glioblastoma stem cells (CSC) have been reported to be sensitive to the mechanical properties of the surrounding tissue/microenvironment and to use the microenvironment stiffness to enhance invasion.In this context, we developed a 3D artificial fibrillary tissue which can allow in vitro recapitulation of the migration behavior observed in vivo. This 3D matrix is highly plastic which allows for modulation of stiffness, surface chemistry and fibers alignment.On the first hand, we functionalized the fibers with extracellular matrix proteins and then we plated GSCs neurospheres (NS) on the developed 3D artificial tissue. The addition of laminin modulates the migration behaviour from single cell to collective mode.On the second hand we have modified stiffness of the fibers. After five days of GSCs NS migration on 3D electrospun fibers of different stiffnesses, we have seen an increase in migration velocity for an intermediate stiffness of 166kPa in comparison with our softest 3.2 kPa and stiffest stiffnesses (1260 kPa). This maximum migration rate is associated with changes in cell shape, increase of EMT proteins expressions and modifications of focal adhesion proteins regulation.Mannoside acetyl glucosaminyltransferase 5 (MGAT5) overexpression is associated with malignant tumors and it is implicated in the clustering of membrane proteins through lattice formation, focal adhesions, cell migration, invasion, and epithelial-to-mesenchymal transition (EMT), resulting in functional advantages for tumor cells.In light of previous results about of EMT proteins expressions and modifications of focal adhesion proteins regulation, we generated MGAT5 CRISPR Cas9 GSCs and placed it on 3D matrix with different stiffnesses. We observe a decrease in migration velocity at the intermediate stiffness in comparison with GSCs WT associated with a decrease in focal adhesion maturation and EMT. This study demonstrates the implication of glycosylation and more particularly MGAT5-mediated glycosylation on GSCs mechanotransduction.Les cellules souches de glioblastomes (GSC) sont sensibles aux propriétés mécaniques de leur microenvironnement et utilisent la rigidité pour favoriser leur invasion.Dans ce contexte, nous avons développé une matrice 3D fibrillaire artificielle permettant de récapituler in vitro les comportements migratoires observés in vivo. Cette matrice étant hautement plastique, nous avons pu moduler la rigidité, la chimie de surface ou encore l'alignement des fibres.Dans un premier temps nous avons modifié leur chimie de surface grâce à l’ajout de protéines de la matrice extracellulaire (MEC) puis déposé des neurosphères (NS) de GSC sur ce tissu artificiel. L’ajout de laminine nous a permis d’observer le passage d’un comportement migratoire collectif à individuel.Dans un deuxième temps nous avons modulé la rigidité des fibres. Après cinq jours de migration des GSC dans différentes conditions de rigidités, nous avons constaté une augmentation de la vitesse de migration à la rigidité intermédiaire de 166kPa par rapport à la condition plus souple de 3,2 kPa et à la condition plus rigides de 1260kPa. Cette capacité migratoire maximale dans nos conditions est associée à des changements de morphologie des GSC, à une augmentation de l'expression des protéines associée à la transition épithélio-mésenchymateuse (EMT) et à une modification de la régulation des protéines des adhérences focales.La surexpression de Mannoside acetyl glucosaminyltransférase 5 (MGAT5) est associée aux tumeurs malignes et est impliquée dans la formation de regroupement de protéines membranaires grâce à la formation d’un treillis. Elle favorise également la maturation des adhérences focales, la migration cellulaire, l'invasion et l’EMT, entraînant ainsi des avantages fonctionnels pour les cellules tumorales. Au vu des résultats précédents sur l'expression des protéines EMT et des modifications de la régulation des protéines des adhérences focales, nous avons généré des GSC n’exprimant plus la MGAT5 grâce à la technique CRISPR Cas9 et les avons placées en NS sur les matrices 3D présentant différentes rigidité. Nous avons alors observé une diminution de la vitesse de migration à 166kPa par rapport aux GSC contrôles, associée à une diminution de l'EMT et de la maturation des adhérences focales. Par conséquent, cette étude démontre l'implication de la glycosylation et plus particulièrement de la glycosylation médiée par la MGAT5 sur la mécanotransduction des GSC

Mechanotransduction of glioblastoma stem cells in a new 3D matrix for cell culture

Les cellules souches de glioblastomes (GSC) sont sensibles aux propriétés mécaniques de leur microenvironnement et utilisent la rigidité pour favoriser leur invasion.Dans ce contexte, nous avons développé une matrice 3D fibrillaire artificielle permettant de récapituler in vitro les comportements migratoires observés in vivo. Cette matrice étant hautement plastique, nous avons pu moduler la rigidité, la chimie de surface ou encore l'alignement des fibres.Dans un premier temps nous avons modifié leur chimie de surface grâce à l’ajout de protéines de la matrice extracellulaire (MEC) puis déposé des neurosphères (NS) de GSC sur ce tissu artificiel. L’ajout de laminine nous a permis d’observer le passage d’un comportement migratoire collectif à individuel.Dans un deuxième temps nous avons modulé la rigidité des fibres. Après cinq jours de migration des GSC dans différentes conditions de rigidités, nous avons constaté une augmentation de la vitesse de migration à la rigidité intermédiaire de 166kPa par rapport à la condition plus souple de 3,2 kPa et à la condition plus rigides de 1260kPa. Cette capacité migratoire maximale dans nos conditions est associée à des changements de morphologie des GSC, à une augmentation de l'expression des protéines associée à la transition épithélio-mésenchymateuse (EMT) et à une modification de la régulation des protéines des adhérences focales.La surexpression de Mannoside acetyl glucosaminyltransférase 5 (MGAT5) est associée aux tumeurs malignes et est impliquée dans la formation de regroupement de protéines membranaires grâce à la formation d’un treillis. Elle favorise également la maturation des adhérences focales, la migration cellulaire, l'invasion et l’EMT, entraînant ainsi des avantages fonctionnels pour les cellules tumorales. Au vu des résultats précédents sur l'expression des protéines EMT et des modifications de la régulation des protéines des adhérences focales, nous avons généré des GSC n’exprimant plus la MGAT5 grâce à la technique CRISPR Cas9 et les avons placées en NS sur les matrices 3D présentant différentes rigidité. Nous avons alors observé une diminution de la vitesse de migration à 166kPa par rapport aux GSC contrôles, associée à une diminution de l'EMT et de la maturation des adhérences focales. Par conséquent, cette étude démontre l'implication de la glycosylation et plus particulièrement de la glycosylation médiée par la MGAT5 sur la mécanotransduction des GSC.Glioblastoma stem cells (CSC) have been reported to be sensitive to the mechanical properties of the surrounding tissue/microenvironment and to use the microenvironment stiffness to enhance invasion.In this context, we developed a 3D artificial fibrillary tissue which can allow in vitro recapitulation of the migration behavior observed in vivo. This 3D matrix is highly plastic which allows for modulation of stiffness, surface chemistry and fibers alignment.On the first hand, we functionalized the fibers with extracellular matrix proteins and then we plated GSCs neurospheres (NS) on the developed 3D artificial tissue. The addition of laminin modulates the migration behaviour from single cell to collective mode.On the second hand we have modified stiffness of the fibers. After five days of GSCs NS migration on 3D electrospun fibers of different stiffnesses, we have seen an increase in migration velocity for an intermediate stiffness of 166kPa in comparison with our softest 3.2 kPa and stiffest stiffnesses (1260 kPa). This maximum migration rate is associated with changes in cell shape, increase of EMT proteins expressions and modifications of focal adhesion proteins regulation.Mannoside acetyl glucosaminyltransferase 5 (MGAT5) overexpression is associated with malignant tumors and it is implicated in the clustering of membrane proteins through lattice formation, focal adhesions, cell migration, invasion, and epithelial-to-mesenchymal transition (EMT), resulting in functional advantages for tumor cells.In light of previous results about of EMT proteins expressions and modifications of focal adhesion proteins regulation, we generated MGAT5 CRISPR Cas9 GSCs and placed it on 3D matrix with different stiffnesses. We observe a decrease in migration velocity at the intermediate stiffness in comparison with GSCs WT associated with a decrease in focal adhesion maturation and EMT. This study demonstrates the implication of glycosylation and more particularly MGAT5-mediated glycosylation on GSCs mechanotransduction

A novel 3D nanofibre scaffold conserves the plasticity of glioblastoma stem cell invasion by regulating galectin-3 and integrin-β1 expression

International audienceGlioblastoma Multiforme (GBM) invasiveness renders complete surgical resection impossible and highly invasive Glioblastoma Initiating Cells (GICs) are responsible for tumour recurrence. Their dissemination occurs along pre-existing fibrillary brain structures comprising the aligned myelinated fibres of the corpus callosum (CC) and the laminin (LN)-rich basal lamina of blood vessels. The extracellular matrix (ECM) of these environments regulates GIC migration, but the underlying mechanisms remain largely unknown. In order to recapitulate the composition and the topographic properties of the cerebral ECM in the migration of GICs, we have set up a new aligned polyacrylonitrile (PAN)-derived nanofiber (NF) scaffold. This system is suitable for drug screening as well as discrimination of the migration potential of different glioblastoma stem cells. Functionalisation with LN increases the spatial anisotropy of migration and modulates its mode from collective to single cell migration. Mechanistically, equally similar to what has been observed for mesenchyma I migration of GBM in vivo, is the upregulation of galectin-3 and integrin-beta 1 in Gli4 cells migrating on our NF scaffold. Downregulation of Calpain-2 in GICs migrating in vivo along the CC and in vitro on LN-coated NF underlines a difference in the turnover of focal adhesion (FA) molecules between single-cell and collective types of migration

Lem2 is essential for cardiac development by maintaining nuclear integrity

Aims: Nuclear envelope integrity is essential for the compartmentalization of the nucleus and cytoplasm. Importantly, mutations in genes encoding nuclear envelope (NE) and associated proteins are the second highest cause of familial dilated cardiomyopathy. One such NE protein that causes cardiomyopathy in humans and affects mouse heart development is Lem2. However, its role in the heart remains poorly understood. Methods and results: We generated mice in which Lem2 was specifically ablated either in embryonic cardiomyocytes (Lem2 cKO) or in adult cardiomyocytes (Lem2 iCKO) and carried out detailed physiological, tissue, and cellular analyses. High-resolution episcopic microscopy was used for three-dimensional reconstructions and detailed morphological analyses. RNA-sequencing and immunofluorescence identified altered pathways and cellular phenotypes, and cardiomyocytes were isolated to interrogate nuclear integrity in more detail. In addition, echocardiography provided a physiological assessment of Lem2 iCKO adult mice. We found that Lem2 was essential for cardiac development, and hearts from Lem2 cKO mice were morphologically and transcriptionally underdeveloped. Lem2 cKO hearts displayed high levels of DNA damage, nuclear rupture, and apoptosis. Crucially, we found that these defects were driven by muscle contraction as they were ameliorated by inhibiting myosin contraction and L-type calcium channels. Conversely, reducing Lem2 levels to ∼45% in adult cardiomyocytes did not lead to overt cardiac dysfunction up to 18 months of age. Conclusions: Our data suggest that Lem2 is critical for integrity at the nascent NE in foetal hearts, and protects the nucleus from the mechanical forces of muscle contraction. In contrast, the adult heart is not detectably affected by partial Lem2 depletion, perhaps owing to a more established NE and increased adaptation to mechanical stress. Taken together, these data provide insights into mechanisms underlying cardiomyopathy in patients with mutations in Lem2 and cardio-laminopathies in general