Visualizing the spatiotemporal map of Rac activation in bovine aortic endothelial cells under laminar and disturbed flows.

Shuai Shao,Cheng Xiang,Xiaoling Liao,Aziz Ur Rehman Aziz,Kairong Qin,Bo Liu,Juan Carlos Del Alamo

doi:10.1371/journal.pone.0189088

Abstract

Disturbed flow can eliminate the alignment of endothelial cells in the direction of laminar flow, and significantly impacts on atherosclerosis in collateral arteries near the bifurcation and high curvature regions. While shear stress induced Rac polarity has been shown to play crucial roles in cell polarity and migration, little is known about the spatiotemporal map of Rac under disturbed flow, and the mechanism of flow-induced cell polarity still needs to be elucidated. In this paper, disturbed flow or laminar flow with 15 dyn/cm2 of average shear stress was applied on bovine aortic endothelial cells (BAECs) for 30 minutes. A genetically-encoded PAK-PBD-GFP reporter was transfected into BAECs to visualize the real-time activation of Rac in living cell under fluorescence microscope. The imaging of the fluorescence intensity was analyzed by Matlab and the normalized data was converted into 3D spatiotemporal map. Then the changes of data upon chemical interference were fitted with logistic curve to explore the rule and mechanism of Rac polarity under laminar or disturbed flow. A polarized Rac activation was observed at the downstream edge along the laminar flow, which was enhanced by benzol alcohol-enhanced membrane fluidity but inhibited by nocodazole-disrupted microtubules or cholesterol-inhibited membrane fluidity, while no obvious polarized Rac activation could be found upon disturbed flow application. It is concluded that disturbed flow inhibits the flow-induced Rac polarized activation, which is related to the interaction of cell membrane and cytoskeleton, especially the microtubules.

Highlights

Atherosclerosis occurs at the branch points and curved regions of the arterial tree, where the blood flow remains unsteady and the shear stress shows marked spatial and temporal variations [1]
The fluorescent probe used in our experiment was a genetically-encoded PAK-PBD-GFP reporter containing a PAK domain to bind to active Rac and transfected into Bovine aortic endothelial cells (BAECs) to obverse the real-time translocation of active Rac in living cell[13,14,15]
Rac could not be activated in a polarized way upon disturbed flow, and the polarity of Rac should be related to the exact flow direction on the cell surface under flow

Summary

Introduction

Atherosclerosis occurs at the branch points and curved regions of the arterial tree, where the blood flow remains unsteady and the shear stress shows marked spatial and temporal variations [1]. This should be due to the variations of flow patterns-induced functional differences of endothelial cells (ECs), such as migration and proliferation, in vascular system [2]. ECs orient prominently parallel to the direction of blood flow in the straight part of the arterial tree, while little orientation of ECs is found at the branch points and curved regions where flow patterns are more disturbed with no clear forward direction [3]. Laminar fluid shear would cause cell deformation along the flow direction [4], while Bovine aortic endothelial cells (BAECs) subjected to disturbed flow have a morphology and random orientations similar to those under static condition [5]

Methods

Results

Conclusion