Browsing by Author "Gerhardt, Holger"
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- A 96-wells fluidic system for high-throughput screenings under laminar high wall shear stress conditionsPublication . Fonseca, Catarina Gonçalves; Silvério, Vânia; Barata, David; Giese, Wolfgang; Gerhardt, Holger; Cardoso, Susana; Franco, Cláudio AreiasThe ability of endothelial cells to respond to blood flow is fundamental for the correct formation and maintenance of a functional and hierarchically organized vascular network. Defective flow responses, in particular related to high flow conditions, have been associated with atherosclerosis, stroke, arteriovenous malformations, and neurodegenerative diseases. Yet, the molecular mechanisms involved in high flow response are still poorly understood. Here, we described the development and validation of a 96-wells fluidic system, with interchangeable cell culture and fluidics, to perform high-throughput screenings under laminar high-flow conditions. We demonstrated that endothelial cells in our newly developed 96-wells fluidic system respond to fluid flow-induced shear stress by aligning along the flow direction and increasing the levels of KLF2 and KLF4. We further demonstrate that our 96-wells fluidic system allows for efficient gene knock-down compatible with automated liquid handling for high-throughput screening platforms. Overall, we propose that this modular 96-well fluidic system is an excellent platform to perform genome-wide and/or drug screenings to identify the molecular mechanisms involved in the responses of endothelial cells to high wall shear stress. [Figure not available: see fulltext.].
- Tyrosine-protein kinase Yes controls endothelial junctional plasticity and barrier integrity by regulating VE-cadherin phosphorylation and endocytosisPublication . Jin, Yi; Ding, Yindi; Richards, Mark; Kaakinen, Mika; Giese, Wolfgang; Baumann, Elisabeth; Szymborska, Anna; Rosa, André; Nordling, Sofia; Schimmel, Lilian; Akmeriç, Emir Bora; Pena, Andreia; Nwadozi, Emmanuel; Jamalpour, Maria; Holstein, Katrin; Sáinz-Jaspeado, Miguel; Bernabeu, Miguel O.; Welsh, Michael; Gordon, Emma; Franco, Claudio A.; Vestweber, Dietmar; Eklund, Lauri; Gerhardt, Holger; Claesson-Welsh, LenaVascular endothelial (VE)-cadherin in endothelial adherens junctions is an essential component of the vascular barrier, critical for tissue homeostasis and implicated in diseases such as cancer and retinopathies. Inhibitors of Src cytoplasmic tyrosine kinase have been applied to suppress VE-cadherin tyrosine phosphorylation and prevent excessive leakage, edema and high interstitial pressure. Here we show that the Src-related Yes tyrosine kinase, rather than Src, is localized at endothelial cell (EC) junctions where it becomes activated in a flow-dependent manner. EC-specific Yes1 deletion suppresses VE-cadherin phosphorylation and arrests VE-cadherin at EC junctions. This is accompanied by loss of EC collective migration and exaggerated agonist-induced macromolecular leakage. Overexpression of Yes1 causes ectopic VE-cadherin phosphorylation, while vascular leakage is unaffected. In contrast, in EC-specific Src deficiency, VE-cadherin internalization is maintained and leakage is suppressed. In conclusion, Yes-mediated phosphorylation regulates constitutive VE-cadherin turnover, thereby maintaining endothelial junction plasticity and vascular integrity.