The Role of Laminar Fluid Shear Stress on the Morphology, Motility, and Biochemical Expression of Brain Microvascular Endothelial Cells
Reinitz, Adam D.
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To determine the effect of laminar fluid shear stress on the morphology, motility, and biochemical expression of human brain microvascular endothelial cells (HBMEC), we designed a microfluidic flow system to allow live-cell, time lapse imaging of a monolayer of endothelial cells being exposed to physiological levels of shear stress (τ). Two cell lines were tested; HBMECs and human umbilical vein endothelial cells (HUVEC). We quantified the morphological response based on inverse aspect ratio (IAR), the ratio of the minor and major axes of a cell, and orientation, the angle (0-90o) between the major axis of the cell and the direction of flow. Time-lapse imaging allowed for determination of time-dependent changes in morphological parameters and cell motility. After 36h of flow, the HBMECs had orientations of 46o ± .02, 47o± .4, and 47o ± .6 and IAR values of 0.654 ± .007, 0.650 ± .006, and 0.658 ± .005 at 8, 12, and 16 dyn cm-2, respectively. After 36h of flow, the HUVECs had orientations of 43o ± 3o, 36o ± 3o, and 31o ± 2o and IAR values of 0.60 ± .03, 0.58 ± .02, and 0.54 ± .03 at 8, 12, and 16 dyn cm-2, respectively. Time-lapse videos showed significant HBMEC proliferation, as well as delamination at high shear stress. Both cell lines showed a transient motility response, experiencing a rapid increase in motility at the onset of flow, followed by a gradual decline to a steady state condition that was lower than the starting motility. Fluorescence staining showed actin stress fibers in HBMECs not restructuring significantly as compared to the HUVECs. Quantification of F-actin orientation indicated that the HBMEC actin network was randomly oriented, while the HUVECs showed preferential orientation with the direction of flow. Biochemical expression was evaluated using polymerase chain reaction (PCR). Preliminary results indicate upregulation of the junctional proteins claudin-5 and β-catenin. The results suggest that the HBMECs do not have the same mechanisms for mechanical transduction as is seen in other parts of the vasculature. Additionally, the results indicate that in the absence of other physiological interactions, the HBMECs do not adhere tightly to the basement membrane and show minimal contact inhibition of cell growth.