Heparan Sulfate Proteoglycan Is a Mechanosensor on Endothelial Cells

Abstract

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 role of endothelial glycocalyx components in mechanotransduction of fluid shear stress

“…In the present investigation, we extend the in vitro study of Florian et al [9] by exposing BAECs to shear stress after treatment with heparinase, chondroitinase, hyaluronidase, and neuraminidase to selectively degrade key components of the glycocalyx (HS, CS, HA, SA, respectively). In addition to measuring NO production, we also measure the shear-induced production of prostacyclin (PGI 2 ).…”

“…Several studies in live animals or with intact vessels showed that depletion of SA [7,8] or HA (19)with enzymes abolished flow-dependent vasodilation or nitric oxide (NO) production. Florian et al [9] presented the first in vitro evidence that GAGs may mediate EC mechanotransduction. They used a heparinase enzyme to selectively degrade the HS component of bovine aortic endothelial cells (BAECs), and observed that the substantial production of NO induced by steady or oscillatory shear stress could be completely inhibited by a dose of enzyme that removed only 46% of the fluorescence intensity associated with a HS antibody.…”

“…They used a heparinase enzyme to selectively degrade the HS component of bovine aortic endothelial cells (BAECs), and observed that the substantial production of NO induced by steady or oscillatory shear stress could be completely inhibited by a dose of enzyme that removed only 46% of the fluorescence intensity associated with a HS antibody. Thi et al [10] exposed monolayers of rat fat-pad ECs to steady shear stress and showed that the characteristic reorganization of peripheral actin bands in response to shear was completely blocked when protein was removed from the media or when protein was present but the monolayer was treated with heparinase as in [9].…”

Primary cilia sensitize endothelial cells for fluid shear stress

“…Hyaluronic acid is also the major component of glycocalyx, which is a hydrated proteoglycan layer covering the luminal aspect of EC Reitsma et al, 2007). Interestingly, glycocalyx production by EC is related to fluid shear stress and to atherosclerosis in hypercholesterolemic and hyperglycaemic patients (Wein- baum et Florian et al, 2003;van den Berg et al, 2006;Nieuwdorp et al, 2006). CD31 did not respond to fluid shear stress and served as a negative control.…”

Change in shear stress (Δτ)/hydraulic conductivity (Lp) relationship after pronase treatment of individual capillaries in situ

“…According to data from Mochizuki et al (Mochizuki et al, 2003; isolated canine femoral arteries) and Florian et al (Florian et al, 2003; cultured endothelial cells) disrupting components of the glycocalyx enzymatically prevented nitric oxide release. It would follow that if nitric oxide is released similarly in frog mesenteric capillaries, then the increased L p after pronase implies an antagonistic role for nitric oxide with regard to barrier function.…”