Fluid shear stress is an important mediator of vascular permeability, yet the molecular mechanisms underlying the effect of shear on the blood‐brain barrier (BBB) have yet to be clarified in cerebral vasculature despite its importance for brain homeostasis. The goal of this study is to probe components of shear mechanotransduction within the BBB to gain a better understanding of pathologies associated with changes in cerebral perfusion including ischemic stroke. Interrogating the effects of shear stress in vivo is complicated by the complexity of factors in the brain parenchyma and the difficulty associated with modulating blood flow regimes. The in vitro model used in this study is compatible with real‐time measurement of barrier function using a transendothelial electrical resistance as well as immunocytochemistry and dextran permeability assays. These experiments reveal that there is a threshold level of shear stress required for barrier formation and that the composition of the extracellular matrix, specifically the presence of high molecular weight hyaluronan, dictates the flow response. Gene editing to modulate the expression of CD44, a mechanosensitive receptor for hyaluronan, demonstrates that the receptor is required for the endothelial response to shear stress. Manipulation of small GTPase activity reveals CD44 activates Rac1 while inhibiting RhoA activation. Additionally, adducin‐γ localizes to tight junctions in response to shear stress and RhoA inhibition and is required to maintain the barrier. This study identifies specific components of the mechanosensing complex associated with the BBB response to fluid shear stress and, therefore, illuminates potential targets for barrier manipulation in vivo.

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