(489f) Effects of Oscillatory Fluid Shear Stress On Endothelial Cellular Responses in the Presence of LTB4 and LPS

Chakraborty, A., University of Louisville
Jala, V. R., University of Louisville
Bodduluri, H., University of Louisville
Berson, R. E., University of Louisville

The role of LPS, (Lypo-polysaccharide), Leukotriene B4 (LTB4) and its receptor (BLT1) are implicated in several inflammatory diseases including asthma, arthritis, and atherosclerosis. LPS has been shown to induce BLT1 expression up to 10-fold in HUVECs compared to normal cells. Wall shear stresses (WSS) are commonly accepted as the primary influence affecting anchored cells subjected to fluid flow. Therefore, it is necessary to analyze the shear effects on cells in the presence of LTB4 and LPS. Culture dishes on orbiting platforms are commonly used for in vitro cell culture studies. One advantage is that they allow for tens to hundreds of cases to be studied at once since dishes can be stacked on the platform. The fluid flow in such systems is oscillatory in nature due to a travelling waveform with different WSS magnitudes at different locations on the bottom surface of the dish. A novel technique has been applied here by which endothelial cellular responses in the presence of LTB4 and LPS can be analyzed under oscillating shear exposure at different locations in the dish in vitro. Determination of shear has been accomplished using Computational Fluid Dynamics (CFD) to determine the magnitude of oscillating WSS exerted by fluid flow. With CFD software, a model has been created that provides complete temporal and spatial resolution of exerted shear on the bottom of the dish where anchored cells reside. For instance, at an orbital speed of 100 rpm, the shear range near the edge of the dish oscillates between 0-9 dyne/cm2 whereas the range near the center oscillates between 0-3 dyne/cm2.

It has been shown that shear stress induces several genes which play a critical role in the development of several inflammatory diseases. The effect of oscillating shear on atherogenic genes (BLT1, CCL2, TNF-alfa, IFN-gama and NFK-beta) has been analyzed by measuring mRNA levels at different locations of the dish under different magnitudes of oscillating shear. This has been done by the Real Time PCR method. Interestingly, results showed that the shear affected atherogenic genes behave differently when compared to cells not subjected to shear, and gene expression differs depending on the location in the dish. Expression levels of BLT1 in HUVECs cells have been determined by flow cytometry and compared with normal cells at different locations (0.5 cm, 1.5 cm and 2.1 cm from the center of the dish). BLT1 expression levels for cells with LTB4 and LPS are seen to be affected differently by shear exposure at these different locations. Furthermore, cell proliferation is also seen to vary in the presence of LTB4 and LPS and also vary based on location in the dish as compared to cells in stationary culture.