This review describes a novel, easy to operate platform to investigate the effect of unidirectional or bidirectional fluid flow shear stress on endothelial cells. It further uses the system to investigate the effect of flow on endothelial cell orientation, actin organization, and Golgi-polarization in both wildtype and TIE2 negative endothelial cells. The manuscript is of interest to the field, overall sound and clearly written. The following minor remarks could be addressed to further enhance the manuscript:

• Page 5, methods volume flow measurement: the formula presented at the end of the paragraph does not give the flow rate at a certain timepoint, but the average flowrate from the start of the experiment until the timepoint t. A better formula would be Qt = (Vt – Vt-1) / (t – (t-1)) (so volume at current timepoint minus volume at previous timepoint divided by the time between the current and the previous timepoint).
• Page 9 midway paragraph; it is clear that flow through the channel will be very low when the bypass is active using a right tilt. However, it is not directly clear that there is no flow at all (leading to true unidirectional flow). It would be nice to show for instance a computational analysis of the actual flow / wall shear stress that can be expected in this situation.
• Figure 1 page 11: a description of panel ‘d’ is missing in the figure legend
• Figure 2 page 12: for panels ‘d’ and ‘e’ use either Time (s) or Time (min) for both
• Figure 2 page 12: for panel ‘f’ it would be informative to also include the profiles at other timepoints (for instance 60, 120 and 180 seconds) to get a better idea of how the shear stresses vary during the draining of the reservoir.
• Figure 2 page 12: in panel ‘d’, the graph of the volume over time appears to be rather linear, which would correspond with a constant value for the flow rate (the slope of the volume plotted vs time). Yet, in panel ‘e’, a very clear decline in the flow rate over time is seen. Please address this apparent discrepancy.
• Page 12 2nd and 3rd sentence: references to Fig. 2d and 2e appear to be switched
• Page 18 midway: elongation is shown in 5h and 5k, no in 5i
• Figure 7 page 23: please describe the meaning of the dotted red lines in panel d in the figure legend.

The authors have carefully addressed the comments, resulting in an improved manuscript. In my opinion, the current manuscript can be accepted for publication.