Abstract 16917: Shear Stress Regulates Blood-Brain Barrier Integrity Through the CD44 Receptor

Abstract

Rationale: The “no-reflow” period that follows a stroke is characterized by reduced blood flow and an increase in blood-brain barrier (BBB) permeability, which exacerbates injury to the surrounding tissue. Objective: Here, we evaluate the hypothesis that reduced shear stress on the endothelium contributes to BBB breakdown through attenuation of a cd44-mediated signaling mechanism. Methods and Results: Our studies rely on a three-dimensional in vitro model of a cerebral arteriole that mimics specific aspects of the in vivo BBB, including an endothelium featuring tight junctions, a robust basement membrane, and interaction with astrocytic endfeet. The in vitro vessels are patterned inside a collagen/hyaluronan (HA) composite scaffold within a microfluidic device that allows for the application of controlled levels of shear stress to the endothelium. Perfusion with culture medium exerting a shear stress of at least 0.07 Pa for a period of four days establishes vessel permeability on par with in vivo vasculature, as determined by dextran exclusion experiments and transendothelial electrical resistance (TEER) using impedance spectroscopy. Ceasing the fluid flow resulted in a significant increase in the permeability of the in vitro vessels within two hours (Figure 1A). Removal of HA from the scaffold prevents the formation of tight junctions, suggesting that a cell surface receptor specific for HA could be responsible for the mechanotransduction of fluid shear stress. Knockout of the cd44 receptor using CRISPR-Cas9 negated flow-mediated barrier formation, as shown by reduced tight junction formation in Figure 1B. Quantification of vessel permeability demonstrated that cd44 knockout had a significant affect on barrier function (Figure 1C). Conclusions: Our in vitro model demonstrates the importance of shear stress for maintaining the BBB. Moreover, our findings indicate that the cd44 receptor is a crucial component of the mechanosensing complex that mediates the endothelial response to shear stress. Figure 1: A) Vessel permeability following cessation of fluid flow. B) 180-um diameter cd44 KO endothelial channel (green: actin, red: zonula-occludin 1, blue: nuclei). C) the effect of HA and cd44 knockout on vessel permeability.