New insights into shear stress-induced endothelial signalling and barrier function: cell-free fluid versus blood flow.
OBJECTIVE
Fluid shear stress (SS) is known to regulate endothelial cell (EC) function. Most of the studies, however, focused on the effects of cell-free fluid-generated wall SS on ECs. The objective of this study was to investigate how changes in blood flow altered EC signalling and endothelial function directly through wall SS and indirectly through SS effects on red blood cells (RBCs).


RESULTS
Experiments were conducted in individually perfused rat venules. We experimentally induced changes in SS that were quantified by measured flow velocity and fluid viscosity. The concomitant changes in EC [Ca2+]i and nitric oxide (NO) were measured with fluorescent markers, and EC barrier function was assessed by fluorescent microsphere accumulation at EC junctions using confocal imaging. EC eNOS activation was evaluated by immunostaining. In response to changes in SS, increases in EC [Ca2+]i and gap formation occurred only in blood or RBC solution perfused vessels, whereas SS-dependent NO production and eNOS-Ser1177 phosphorylation occurred in both plasma and blood perfused vessels. A bioluminescent assay detected SS-dependent ATP release from RBCs. Pharmacological inhibition and genetic modification of pannexin-1 channels on RBCs abolished SS-dependent ATP release and SS-induced increases in EC [Ca2+]i and gap formation.


CONCLUSIONS
SS-induced EC NO production occurs in both cell free fluid and blood perfused vessels, whereas SS-induced increases in EC [Ca2+]i and EC gap formation require the presence of RBCs, attributing to SS-induced pannexin-1 channel dependent release of ATP from RBCs. Thus, changes in blood flow alter vascular EC function through both wall SS and SS exerted on RBCs, and RBC released ATP contributes to SS-induced changes in EC barrier function.
Department of Cellular and Molecular Physiology, College of Medicine, Pennsylvania University, 500 University Drive, Hershey, PA 17033, USA.
Sulei
S
Xu
Department of Cellular and Molecular Physiology, College of Medicine, Pennsylvania University, 500 University Drive, Hershey, PA 17033, USA.
Xiang
X
Li
Department of Cellular and Molecular Physiology, College of Medicine, Pennsylvania University, 500 University Drive, Hershey, PA 17033, USA.
Kyle Brian
KB
LaPenna
Department of Physiology and Pharmacology, School of Medicine, West Virginia University, One Medical Center Drive, Morgantown, WV 26506, USA.
Stanley David
SD
Yokota
Department of Medicine, University of Alabama at Birmingham, 901 19th street South. Birmingham, AL 35294, USA.
Sabine
S
Huke
Department of Cellular and Molecular Physiology, College of Medicine, Pennsylvania University, 500 University Drive, Hershey, PA 17033, USA.
Pingnian
P
He
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001783:Blood Flow Velocity; D001809:Blood Viscosity; D002118:Calcium / Q000378:metabolism; D002199:Capillary Permeability; D042783:Endothelial Cells / Q000378:metabolism*; D004789:Enzyme Activation; D004912:Erythrocytes / Q000378:metabolism*; D005260:Female; D017629:Gap Junctions / Q000378:metabolism; D066298:In Vitro Techniques; D040542:Mechanotransduction, Cellular; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010477:Perfusion; D010766:Phosphorylation; D017207:Rats, Sprague-Dawley; D012039:Regional Blood Flow; D013314:Stress, Mechanical; D013997:Time Factors; D014699:Venules / Q000378:metabolism*
D009569:Nitric Oxide; D000255:Adenosine Triphosphate; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D002118:Calcium
ATP; Endothelial calcium; Microvessel permeability; Nitric oxide; Shear stress