Fig. 5

Gamma-secretase inhibitors (GSI) modulate oscillatory shear-induced EndMT and cell invasiveness. A, B Inhibition of Notch receptor cleavage by 500 nM DAPT and 250 nM RO4929097 prevented the overexpression of SNAI1/2 and N-cadherin in hCMEC/d3 exposed to oscillatory flow for 24 h. Integrin α9/β1 expression was augmented by GSI even in the continuing presence of oscillatory shear stress. EndMT-associated molecular changes were significantly reduced in the presence of RO4929097 (scale bar 20 µM, magnification ×40). C Fluorescent microscopic images of invaded cerebral microvascular endothelial cells, with prior exposure to control and oscillatory flows, at the lower surface of the transwell Matrigel-coated filter membrane stained with 5 μg/ml of nuclear stain Hoechst 33342 in the presence and absence of each inhibitor (250 nM RO4929097 and 500 nM DAPT) (scale bar 100 µM, magnification ×10). D Invasion assay was done in triplicate, and invaded cells were counted by ImageJ and plotted graphically. As noted in the graph, cells exposed to 24 h oscillatory flow invade faster than normal endothelial cells. The presence of DAPT (p < 0.001) and RO4929097 (p < 0.0001) effectively reduced the percentage of invaded oscillatory shear-exposed endothelial cells when compared with cells exposed to oscillatory flow alone. OF represents oscillatory shear stress. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus respective static or parallel uniform shear-treated groups