Alterations in the composition of the glycocalyx of venular endothelium in postcapillary venules (rat mesentery) were explored in models of inflammation and ischemia-reperfusion injury. Lectins were covalently linked to fluorescently labeled microspheres (0.1-microm diameter) or directly labeled with FITC. Adhesion of lectins specific for glucose and galactose residues of glycosaminoglycans (GAGs) and other components of the endothelial glycocalyx decreased dramatically after superfusion of the mesentery with the chemoattractant N-formylmethionyl-leucyl-phenylalanine and during reperfusion after 60-min ischemia. These reductions were significantly attenuated by superfusion with pertussis toxin (PTX), suggesting that shedding of glycocalyx was mediated by G proteins. Adhesion of microspheres linked with antibody for syndecan-1, a major proteoglycan to which GAGs are bound, revealed increased labeling as GAGs were lost and permitted greater numbers of spheres to adhere to the protein core, which was not shed. Induction of ischemia by occluding proximal microvessels for 60 min resulted in a 40% increase in galactosaminoglycans and a 15% increase in glucosaminoglycans on the endothelium, which was not inhibited by PTX. Reperfusion of vessels led to a rapid loss of GAGs that was inhibited by pretreatment with PTX, with 40% of galactosaminoglycans and 25% of glucosaminoglycans accumulated being removed by G protein-mediated shedding and the remainder freely convected away by fluid shear. We conclude that the composition of the glycocalyx results from a balance of the rate of biosynthesis of GAGs by the endothelial cell and their shedding, which may be mediated by intracellular and/or membrane-bound proteases or lyases released or activated by G protein signaling.

The goal of elucidating the biophysical and physiological basis of pressure-flow relations in the microcirculation has been a recurring theme since the first observations of capillary blood flow in living tissues. At the birth of the Microcirculatory Society, seminal observations on the heterogeneous distribution of blood cells in the microvasculature and the rheological properties of blood in small bore tubes raised many questions on the viscous properties of blood flow in the microcirculation that captured the attention of the Society's membership. It is now recognized that blood viscosity in small bore tubes may fall dramatically as shear rates are increased, and increase (dramatically with elevations in hematocrit. These relationships are strongly affected by blood cell deformability and concentration, red cell aggregation, and white cell interactions with the red cells anti endothelium. Increasing strength of red cell aggregation may result in sequestration of clumps of red cells with either reductions or increases in microvascular hematocrit dependent upon network topography. During red cell aggregation, resistance to flow may thus decrease with hematocrit reduction or increase due to redistribution of red cells. Blood cell adhesion to the microvessel wall may initiate flow reductions, as, for example, in the case of red cell adhesion to the endothelium in sickle cell disease, or leukocyte adhesion in inflammation. The endothelial glycocalyx has been shown to result from a balance of the biosynthesis of new glycans, and the enzymatic or shear-dependent alterations in its composition. Flow-dependent reductions in the endothelial surface layer may thus affect the resistance to flow and/or the adhesion of red cells and/or leukocytes to the endothelium. Thus, future studies aimed at the molecular rheology of the endothelial surface layer may provide new insights into determinants of the resistance to flow.

The binding of fluorescently labeled microspheres (FLMs, 0.1-microm diameter) coated with antibody (1a29) to ICAM-1 was studied in postcapillary venules during topical application of the chemoattractant N-formylmethionyl-leucyl-phenylalanine (fMLP). FLM adhesion to endothelial cells (ECs) increased dramatically from 50 to 150 spheres per 100-microm length of venule after superfusion of the mesentery with fMLP and equaled or exceeded levels of leukocyte (WBC) adhesion. Removal of the EC glycocalyx by micropipette infusion of the venule with heparinase increased FLM-EC adhesion to levels attained with fMLP. Subsequent application of fMLP did not increase FLM adhesion further, suggesting that the FLMs saturated all ICAM-1 binding sites. Perfusion with heparinase after suffusion with fMLP significantly increased FLM-EC adhesion above levels attained with fMLP. However, WBC adhesion fell because of possible removal of selectins necessary to maintain WBC rolling at the wall. It is concluded that the glycocalyx serves as a barrier to adhesion and that its shedding during natural activation of ECs may be an essential part of the inflammatory response.

In vivo studies of the rheological behavior of blood in the microcirculation were conducted by direct in situ measurements in cat mesentery. Upstream to downstream pressure drops were measured in unbranched arterioles, capillaries, and venules, with diameters from 7 to 58 micrometer. Simultaneous measurements of red cell velocity and vessel geometry facilitated computation of bulk velocity, pressure gradient, apparent viscosity, wall shear stress, and resistance. Arteriovenous distributions of these parameters revealed the following. Maximum pressure gradient (0.015 cm H20/micrometer) occurs in the true capillaries (7 micrometer in diameter); intravascular wall shear stress averaged 47.1 dynes/cm2 in arterioles and 29.0 dynes/cm2 in venules. Extreme values as great as 200 dynes/cm2 were observed in a few shunting arterioles. Apparent viscosity averaged 3.59 cP in arterioles, 5.15 cP in venules, and 4.22 cP overall. Intravascular resistance per unit length of microvessel varied with luminal diameter as a power law function with exponents of -4.04 for arterioles, -3.94 for venules, and -3.99 for all vessels combined. This apparent maintenance of Poiseuille's law is attributed to the opposing processes of hematocrit reduction and decreasing shear rate as blood is dispersed in successive arteriolar segments, and the converse action of these processes in the venous confluences which lessen the extent of network variations in apparent viscosity. Reductions in bulk velocity from the normal flow state to below 0.5 mm/sec resulted in increases in apparent viscosity by a factor of 2 to 10, which are attributed primarily to obstruction of the lumen by leukocyte-endothelium adhesion.

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