Relative shedding of glycosaminoglycans from the endothelial glycocalyx during inflammation and their contribution to stiffness of the glycocalyx.

Abstract

BACKGROUND The endothelial (EC) surface layer (glycocalyx) has been shown to act as a barrier to transvascular exchange of solutes, and adhesion of leukocytes (WBCs) during the inflammatory process. It is a labile structure whose components are readily shed by the action of proteases and endoglycosidases. Details of shedding of specific constituents of the glycocalyx remain to be determined. OBJECTIVES To review the contributions of the primary glycosaminoglycans that comprise the glycocalyx, heparan sulfate (HS), chondroitin sulfate (CS) and hyaluronan (HA), as barrier to WBC-EC adhesion, and elucidate the rates of shedding of each component in response to an inflammatory stimulus. Assess the potential role that stiffness of the glycocalyx plays in resisting infiltration by WBCs during the adhesion process. METHODS Quantitate shedding of the glycocalyx in post-capillary venules of rat mesentery in response to superfusion of the tissue with 10-6 M fMLP. The presence and loss of HS, CS and HA was assessed by labeling all components with fluorescently labelled lectin (BS-1) or HS antibodies, and HA with fluorescently labelled hyaluronan binding protein (HBP). RESULTS Following a 30 min exposure of the mesentery to fMLP about 50% of HBP was lost in contrast to a previously shown loss of 20% of lectin labelled GAGs, and 25% loss of Mab labelled HS. The time constant for HBP shedding (5.8 min) was one-third that for BS-1 labelled GAGs (14.3 min). An attempt was made to assess stiffness of the glycocalyx by observing the motion of adhered lectin coated fluorescently labelled microspheres (FLM) under oscillatory flow conditions. Estimates of the elastic modulus of the glycocalyx revealed a value of 26 mPa, which was orders of magnitude below published data obtained by atomic force microscopy. CONCLUSIONS The relatively rapid shedding of HA compared to HS was consistent with the hypothesis that HA may form the dominant barrier to WBC-EC adhesion. Prior observations that HA lies closer to and parallel to the endothelial membrane, compared to HS suggests that the compact layer of HA near the EC membrane surrounds WBC adhesion receptors that are much shorter in length than the total thickness of the glycocalyx. The relatively low elastic modulus of the glycocalyx under shear is consistent with the hypothesis that the FLMs adhered to strands of HS normal to the EC surface that extended above the relatively more compact and stiffer HA layer below. Gradients of stiffness within the glycocalyx may not be detected by compressive indentation tests published to date.