Abstract-Previous studies have demonstrated the relationship of occludin expression to endothelial layer permeability; but none have determined the relationship between prolonged exposure to shear stress and occludin expression. The purpose of this study is to determine the effects of varying fluid shear stress on the regulation of occludin in endothelial cells after cell realignment. A flow system, which consisted of four parallel plate flow chambers, was developed in order to simultaneously expose human umbilical vein endothelial cells to 0.3, 10, 20, and 30 dynes/cm 2 levels of shear stress. A constant flow rate was applied to all flow chambers and shear stress was controlled by varying the viscosities of the flowing media. To date, results have shown that occludin expression is down-regulated, though only by <10% over the entire range, by increases in shear stress magnitude. Occludin expression in the static cultures were also found to be significantly greater than those exposed to shear. These differences show that occludin expression is a shear dependent phenomenon , but not as large a difference was observed as compared to previously reported results for short time exposures . Keywords - endothelial cells, occludin, shear stress I. INTRODUCTION Endothelial cells (ECs) line veins and arteries and are crucial for the maintenance of the solute barrier between the flowing blood and the underlying tissue. Junctions between neighboring ECs impede the passage of solutes through paracellular pathways. The transmembrane protein occludin [1] has been located in the tight junctions where cells are closest to each other, and the cleaving of this protein corresponds to an increase in the permeability of the paracellular pathway. Fluid shear stress has been shown to affect ECs by changing their shape [2] or by regulating the expression of proteins and growth factors [3]. Recently, shear stress and mechanical strain have been found to down- regulate occludin expression in bovine aortic endothelial cells [4] and alveolar epithelial cells [5], respectively.. These experiments observed these effects within 3 hours of the induced strain and did not address realignment issues that are occurring within the first 24 hours of the cells’ exposure. The purpose of this study was to determine the effects of varying fluid shear stress on the expression of occludin in endothelial cells after cells realignment. II. METHODOLOGY Human umbilical vein endothelial cells were isolated using standard techniques. Prior to utilization in the flow study, the cells were cultured in T-75 flasks with Endothelial Growth Media – 2 (Clonetics, Walkersville, MD) through passage three, after which they were cryogenically frozen Figure 1. Schematic of flow chamber base. for individual use. For each experiment, a vial of cells was plated onto a T-75 flask, grown to confluency, and then plated onto fiveglass microscope slides (38 mm x 75 mm) coated with a fibronectin (10 μg/ml) intercellular matrix. When the cells were 80-90% confluent, the slides were connected to the flow chambers for the experiments and grown while subjected to a flowing media in a custom built environmental chamber. The flow chambers were designed using parallel plate theory with the glass slide serving as the bottom plate and the surface of the stainless steel flow chamber base acting as the second plate. The 'plates' were separated by a fiber reinforced silicon gasket (t = 254 μm). The four flow chambers were fabricated with identical geometries, so that four different shear levels could be tested simultaneously by simply adjusting the viscosity of the flowing media. Previous research has found that physiological shear stress levels in arteries range between ~15-24 dynes/cm 2 [6]. Therefore, shear levels of 0.3, 10, 20, and 30 dynes/cm 2 were tested in this study. With a flow rate of 12.3 ml/min, dynamic viscosities of 1.32, 2.64, and 3.96 cP were required to obtain shear stresses of 10, 20, and 30 dynes/cm 2 . The viscosities were increased by adding dextran (MW=39,300) to the media and measured with a cone-and-plate viscometer. The flow was induced by a twelve-roller pump and measured with an ultrasonic flow meter. The EC coated slide was placed in the environmental chamber in a petri dish and allowed to grow under static conditions to act as a control. The EC’s were exposed to shear for thirty hours. Upon completion of the experiments, the slides were inspected for the presence, confluency, and alignment of the ECs using an inverted, phase contrast microscope (Axiovert 100, Zeiss, Munich, Germany). The slides were then stained for occludin first with the primary antibody (Rabbit Anti- Wall Shear Stress Regulation of Occludin Expression in Human Umbilical Vein Endothelial Cells D. M. Brey 1 , W. D. Ehringer 2 , J. S. Alexander 3 , Y. Xu 1 , T. J. Roussel, Jr 1 , R. S. Keynton 1 1 Department of Mechanical Engineering, University of Louisville, Louisville, KY, USA 2 Department of Physiology and Biophysics, University of Louisville, Louisville, KY, USA 3 Department of Molecular and Cellular Physiology, Louisiana State University, Shreveport, LA, USA