Abstract-The objective of this study was to test whether a glycosaminoglycan component of the surface glycocalyx layer is a fluid shear stress sensor on endothelial cells (ECs). Because enhanced nitric oxide (NO) production in response to fluid shear stress is a characteristic and physiologically important response of ECs, we evaluated NO x (NO 2 Ϫ and NO 3 Ϫ ) production in response to fluid shear stress after enzymatic removal of heparan sulfate, the dominant glycosaminoglycan of the EC glycocalyx, from cultured ECs. The significant NO x production induced by steady shear stress (20 dyne/cm 2 ) was inhibited completely by pretreatment with 15 mU/mL heparinase III (E.C.4.2.2.8) for 2 hours. Oscillatory shear stress (10Ϯ15 dyne/cm 2 ) induced an even greater NO x production than steady shear stress that was completely inhibited by pretreatment with heparinase III. Addition of bradykinin (BK) induced significant NO x production that was not inhibited by heparinase pretreatment, demonstrating that the cells were still able to produce abundant NO after heparinase treatment. Fluorescent imaging with a heparan sulfate antibody revealed that heparinase III treatments removed a substantial fraction of the heparan sulfate bound to the surfaces of ECs. In summary, these experiments demonstrate that a heparan sulfate component of the EC glycocalyx participates in mechanosensing that mediates NO production in response to shear stress. The full text of this article is available online at http://www.circresaha.org. Key Words: shear stress Ⅲ endothelial cells Ⅲ heparan sulfate Ⅲ nitric oxide Ⅲ glycocalyx T he inner surfaces of blood vessels are lined with a monolayer of endothelial cells (ECs) that is continually exposed to the mechanical shearing forces (stresses) of blood flow. Variations in shear stress magnitude as well as temporal and spatial distribution have been shown to induce alterations in endothelial permeability and hydraulic conductivity, 1-3 cytoskeletal structure, 4 -7 surface adhesion molecule expression, 8 and gene expression. 9,10 In addition, the exposure of endothelial cells to shear (both steady and oscillatory) has been shown to alter the production of vasoregulating agents of which nitric oxide (NO) is perhaps the most notable. [11][12][13] NO is a vasodilator produced by the conversion of L-arginine to L-citrulline that is catalyzed by endothelial nitric oxide synthase (eNOS). NO modulates vascular tone by eliciting relaxation of smooth muscle cells while inhibiting smooth muscle cell growth. 14 NO production responds to changes in shear stress in a biphasic manner in human umbilical vein endothelial cells (HUVECs) and bovine aortic endothelial cells (BAECs). 11,12 There is an initial rapid NO production phase that is G protein and Ca 2ϩ -dependent and is influenced by rate of change of shear and not the shear level per se. The subsequent phase is characterized by a lower rate of NO production rate that is G protein and Ca 2ϩ -independent but is shear level dependent. 12,15 Both phases of the NO respo...

The surface of endothelial cells is decorated with a wide variety of membrane-bound macromolecules that constitute the glycocalyx. These include glycoproteins bearing acidic oligosaccharides with terminal sialic acids (SA), and proteoglycans with their associated glycosaminoglycan that include: heparan sulfate (HS), chondroitin sulfate (CS) and hyaluronic acid (HA). In this study enzymes were used to selectively degrade glycoclyx components from the surface of bovine aortic endothelial cells and the effects of these alterations on fluid shear-induced nitric oxide (NO) and prostacyclin (PGI 2 ) production were determined. Depletion of HS, HA and SA, but not CS, blocked shear-induced NO production. Surprisingly, the same enzyme depletions that blocked NO production had no influence on shear-induced PGI 2 production. The results may be interpreted in terms of a glypicancaveolae-eNOS mechanism for shear-induced NO transduction, with PGI 2 being transduced in basal adhesion plaques that sense the same reaction stress whether the glycocalyx is intact or not.

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