Yalcin O, Ulker P, Yavuzer U, Meiselman HJ, Baskurt OK. Nitric oxide generation by endothelial cells exposed to shear stress in glass tubes perfused with red blood cell suspensions: role of aggregation. Am J Physiol Heart Circ Physiol 294: H2098-H2105, 2008. First published March 7, 2008 doi:10.1152/ajpheart.00015.2008.-Endothelial function is modulated by wall shear stress acting on the vessel wall, which is determined by fluid velocity and the local viscosity near the vessel wall. Red blood cell (RBC) aggregation may affect the local viscosity by favoring axial migration. The aim of this study was to investigate the role of RBC aggregation, with or without altered plasma viscosity, in the mechanically induced nitric oxide (NO)-related mechanisms of endothelial cells. Human umbilical vein endothelial cells (HUVEC) were cultured on the inner surface of cylindrical glass capillaries that were perfused with RBC suspensions having normal and increased aggregation at a nominal shear stress of 15 dyn/cm 2 . RBC aggregation was enhanced by two different approaches: 1) poloxamer-coated RBC suspended in normal, autologous plasma, resulting in enhanced aggregation but unchanged plasma viscosity and 2) normal RBC suspended in autologous plasma containing 0.5% dextran (mol mass 500 kDa), with a similar level of RBC aggregation but higher plasma viscosity. Compared with normal cells in unmodified plasma, perfusion with suspensions of poloxamer-coated RBC in normal plasma resulted in decreased levels of NO metabolites and serine 1177 phosphorylation of endothelial nitric oxide synthase (eNOS). Perfusion with normal RBC in plasma containing dextran resulted in a NO level that remained elevated, whereas only a modest decrease of phosphorylated eNOS level was observed. The results of this study suggest that increases of RBC aggregation tendency affect endothelial cell functions by altering local blood composition, especially if the alterations of RBC aggregation are due to modified cellular properties and not to plasma composition changes.wall shear stress; human umbilical vein endothelial cells; nitric oxide; endothelial nitric oxide synthase; plasma viscosity IT IS NOW WELL ESTABLISHED that shear forces acting on the vascular wall are one of the main factors regulating endothelial function (11). Endothelial cells regulate their nitric oxide (NO) synthesis in response to altered wall shear stress, with the synthesis playing a key role in vasomotor control (21,39,40,43). The details of mechanotransduction and cellular control mechanisms, including the activation of NO synthesis mechanisms, have been investigated widely and are of current interest (16,17,21). The enzyme endothelial nitric oxide synthase (eNOS) is expressed at a basal level in endothelial cells, with the level of expression modulated depending on the magnitude of local shear forces (21, 39). Additionally, the activity of expressed eNOS is modulated by calcium-dependent and -independent mechanisms through phosphorylation of various domains of the eNOS protein (20...