P2Y2 receptor modulates shear stress-induced cell alignment and actin stress fibers in human umbilical vein endothelial cells

Ramasri Sathanoori,Christa E Müller,Paulina Bryl-Gorecka,Björn Olde,David Erlinge,Gary A Weisman,Laurie Erb

doi:10.1007/s00018-016-2365-0

Ramasri Sathanoori, Christa E Müller + Show 5 more

Open Access

https://doi.org/10.1007/s00018-016-2365-0

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Abstract

Endothelial cells release ATP in response to fluid shear stress, which activates purinergic (P2) receptor-mediated signaling molecules including endothelial nitric oxide (eNOS), a regulator of vascular tone. While P2 receptor-mediated signaling in the vasculature is well studied, the role of P2Y2 receptors in shear stress-associated endothelial cell alignment, cytoskeletal alterations, and wound repair remains ill defined. To address these aspects, human umbilical vein endothelial cell (HUVEC) monolayers were cultured on gelatin-coated dishes and subjected to a shear stress of 1 Pa. HUVECs exposed to either P2Y2 receptor antagonists or siRNA showed impaired fluid shear stress-induced cell alignment, and actin stress fiber formation as early as 6 h. Similarly, when compared to cells expressing the P2Y2 Arg-Gly-Asp (RGD) wild-type receptors, HUVECs transiently expressing the P2Y2 Arg-Gly-Glu (RGE) mutant receptors showed reduced cell alignment and actin stress fiber formation in response to shear stress as well as to P2Y2 receptor agonists in static cultures. Additionally, we observed reduced shear stress-induced phosphorylation of focal adhesion kinase (Y397), and cofilin-1 (S3) with receptor knockdown as well as in cells expressing the P2Y2 RGE mutant receptors. Consistent with the role of P2Y2 receptors in vasodilation, receptor knockdown and overexpression of P2Y2 RGE mutant receptors reduced shear stress-induced phosphorylation of AKT (S473), and eNOS (S1177). Furthermore, in a scratched wound assay, shear stress-induced cell migration was reduced by both pharmacological inhibition and receptor knockdown. Together, our results suggest a novel role for P2Y2 receptor in shear stress-induced cytoskeletal alterations in HUVECs.

Highlights

Mechanical forces such as fluid shear stress directly affect the endothelial cells lining the vessel wall and coordinate the complex events of vasodilation and vascular hemostasis
Transfex was from ATCC; UTP and ATPcS were from Sigma, USA; MRS-2768 was from TOCRIS Bioscience, UK; Cibacron Blue F3GA was from PolySciences, Germany; AR-C118925, the P2Y2 receptor-specific antagonist [27] was from University of Bonn, Germany; Phospho-Stop and complete protease inhibitors were from Roche Life Sciences, USA; Micro BCA protein assay kit was from Thermo Scientific, USA; XT 4–12 % Bis–Tris gel was from BioRad, USA
We observe reduced alignment in human umbilical vein endothelial cell (HUVEC) subjected to 6 h of shear stress in the presence of these antagonists (Fig. 1B) with a majority of cells at random angles and only 3 % of the cells aligned in the direction of flow

Summary

Introduction

Mechanical forces such as fluid shear stress directly affect the endothelial cells lining the vessel wall and coordinate the complex events of vasodilation and vascular hemostasis. These hemodynamic forces play an important role in vascular remodeling, and pathophysiology [1]. The endothelial cells play a critical role in repair following injuries caused due to ischemia, and medical procedures including vein bypass graft [5]. Exposure of venous grafts to arterial biomechanical forces is presumed to be an important stimulus for vascular remodeling and venous arterialization with changes in endothelial cell gene expression [2]

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