Regulation of Cytokine-Mediated Vascular Permeability Under Flow-Induced Shear Stress

Abstract

Endothelial cells form the innermost lining of blood vessels throughout the circulatory system. They exhibit a remarkable ability to adapt rapidly to biomechanical and biochemical stimuli from their microenvironment. Vascular endothelial cells play an essential role during the onset of inflammatory conditions and sepsis. Sepsis accounts for the highest number of mortalities in non-cardiac intensive care units and is linked to numerous other underlying conditions including cancer, inflammatory conditions and diabetes. Cancer patients, in particular, are especially susceptible to infections that lead to sepsis and show significantly higher mortality rates due to the immunocompromised nature of the host defense system. Currently, there are no available treatments for sepsis. Furthermore, TNFα has been implicated as one of the major proinflammatory cytokines in sepsis. In the current work, we used physiologically relevant shear stress rates and translated them into a well-controlled in vitro system applying fluid shear stress onto primary endothelial microvascular endothelial cells. We identified a complex formed by the active form of the small GTPase R-Ras and the cytoskeletal scaffold protein filamin A (FLNa) that can regulate TNFα-mediated activation of endothelial cells under fluid shear stress conditions. R-Ras binds directly to repeat 3 of FLNa forming a complex that is necessary for endothelial barrier integrity. We show here that activated GTP-bound R-Ras blocks vascular permeability. Permeability is monitored using the electrical cell impedance spectroscopy (ECIS) method that acquires real-time transendothelial electrical resistance (TEER) values. From the electrical resistance, impedance and capacitance, endothelial permeability can be derived by employing the ECIS model and quantified at nanoscale precision levels concurrently with endothelial cells subjected to fluid shear stress. We also demonstrate a novel platform comprised of ECIS and physiologically relevant fluid shear stress levels to test novel inhibitors or compounds that block TNFα-mediated vascular permeability. Thus, we show that the R-Ras/FLNa complex is important in regulating vascular endothelial permeability under fluid shear stress conditions. This work may offer insights into the regulation of endothelial permeability by providing novel targets to block vascular hyperpermeability or leakiness.