title: P2Y2receptor modulates shear stress-induced cell alignment and actin stress fibers in human umbilical vein endothelial cells.
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 P2Y2receptors 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 P2Y2receptor 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 P2Y2Arg-Gly-Asp (RGD) wild-type receptors, HUVECs transiently expressing the P2Y2Arg-Gly-Glu (RGE) mutant receptors showed reduced cell alignment and actin stress fiber formation in response to shear stress as well as to P2Y2receptor 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 P2Y2RGE mutant receptors. Consistent with the role of P2Y2receptors in vasodilation, receptor knockdown and overexpression of P2Y2RGE 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 P2Y2receptor in shear stress-induced cytoskeletal alterations in HUVECs.
authors.affiliation: Department of Cardiology, Clinical Sciences, Lund University, Tornavägen 10, BMC-D12, Room D1219b, 221 84, Lund, Sweden. Ramasri.Sathanoori@med.lu.se.
authors.forename: Ramasri
authors.initials: R
authors.lastname: Sathanoori
authors.affiliation: Department of Cardiology, Clinical Sciences, Lund University, Tornavägen 10, BMC-D12, Room D1219b, 221 84, Lund, Sweden.
authors.forename: Paulina
authors.initials: P
authors.lastname: Bryl-Gorecka
authors.affiliation: PharmaCenter, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, Bonn, Germany.
authors.forename: Christa E
authors.initials: CE
authors.lastname: Müller
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia, USA.
authors.forename: Laurie
authors.initials: L
authors.lastname: Erb
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia, USA.
authors.forename: Gary A
authors.initials: GA
authors.lastname: Weisman
authors.affiliation: Department of Cardiology, Clinical Sciences, Lund University, Tornavägen 10, BMC-D12, Room D1219b, 221 84, Lund, Sweden.
authors.forename: Björn
authors.initials: B
authors.lastname: Olde
authors.affiliation: Department of Cardiology, Clinical Sciences, Lund University, Tornavägen 10, BMC-D12, Room D1219b, 221 84, Lund, Sweden.
authors.forename: David
authors.initials: D
authors.lastname: Erlinge
mesh_terms: D000199:Actins / Q000378:metabolism* / Q000648:ultrastructure; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D016023:Integrins / Q000378:metabolism; D009154:Mutation; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010766:Phosphorylation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D058473:Receptors, Purinergic P2Y2 / Q000235:genetics / Q000378:metabolism*; D022502:Stress Fibers / Q000378:metabolism* / Q000648:ultrastructure; D013314:Stress, Mechanical; D014945:Wound Healing
chemical_list: D000199:Actins; D016023:Integrins; D034741:RNA, Small Interfering; D058473:Receptors, Purinergic P2Y2; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III
keywords: FAK; Integrins; P2Y2 receptors; Shear stress; Wound repair