The Impact of Flow on the Nuclear Translocation of NF-kB

In this dissertation, the impact of flow on the nuclear translocation of NF-kB in vascular endothelial cells was investigated. NF-kB is a key promoter of inflammatory responses and its mis-regulation is related to the development of vascular diseases. The aim was to establish a link between hemodynamic forces and NF-kB to gain insight in cardiovascular disease mechanisms such as aneurysms. Human umbilical vein endothelial cells (HUVEC) were transfected with two plasmids: H2B-mCherry and GFP-RelA. The nuclear translocation of NF-kB within the transfected primary cells was verified with TNF-a stimulation and compared to immunohistochemistry of TNF-a stimulated non-transfected cells. In the first part of the thesis, transfected HUVECs were exposed to different flow environments, including uniform low shear stress, uniform high shear stress and a shear stress gradient, and imaged live for 6 hours. Computer vision techniques were applied to track each individual cell and each nuclear NF-kB concentration was evaluated as a function of time. In each experiment, more than 1000 single cells were tracked and analysed. TNF-a stimulation caused a synchronised population response with a nuclear NF-kB peak at 30 minutes. The population mean of cells under static conditions remained constant, while spontaneous nuclear translocation of NF-kB in individual cells was observed. Uniform low shear stress stimulation increased translocating activity after 5 hours of flow. Alternatively, uniform high shear stress promoted increased nuclear translocation, directly after onset of flow and after 5 hours. Small differences of nuclear translocation of NF-kB at different shear stress magnitudes within a shear stress gradient were observed. The percentage of cells experiencing early nuclear translocation increased with increased shear stress. It is believed that high shear stress induces nuclear translocation early, while for low shear stresses, responses are delayed. In the second part of the thesis, a numerical model was developed to predict cell population responses of the NF-kB pathway. The model is deterministic but includes extrinsic noise to mimic stimulus dependent cell-to-cell variability. Population responses to different TNF-a concentrations were predicted in close agreement with live-cell measurements. The model was extended with a shear dependent activation module to predict small variabilities observed in nuclear translocation of NF-kB under different shear conditions. The close agreement between nuclear translocation of NF-kB in a shear stress gradient and the measurements allowed prediction of inflammatory responses in different flow environments such as a backward facing step channel. This work provides a first insight in the temporal dynamics of nuclear translocation of NF-kB in a large population of endothelial cells exposed to different flow environments. Although the effects were much weaker than with TNF-a stimulation, differences between static and flow conditions were observed, which indicate that hemodynamic forces affect intracellular signalling.