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

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.

Role of the Glycocalyx as a Barrier to Leukocyte-Endothelium Adhesion.

Leukocyte (WBC) to endothelial cell (EC) adhesion is a receptor-mediated process governed by the avidity and affinity of selectins, which modulate adhesive forces during WBC rolling, and integrins, which determine the strength of firm adhesion. Adhesion receptors on the EC surface lie below an endothelial surface layer (ESL) comprised of the EC glycocalyx and adsorbed proteins which, in vivo, have a thickness on the order 500 nm. The glycocalyx consists of a matrix of the glycosaminoglycans heparan sulfate and chondroitin sulfate, bound to proteoglycans and encased in hyaluronan.

Correction to: Mesenchymal Stem Cell Deformability and Implications for Microvascular Sequestration.

This article was updated to correct the spelling of author Brittany L. Banik's name.

Mesenchymal Stem Cell Deformability and Implications for Microvascular Sequestration.

Mesenchymal stem cells (MSCs) have received considerable attention in regenerative medicine, particularly in light of prospects for targeted delivery by intra-arterial injection. However, little is known about the mechanics of MSC sequestration in the microvasculature and the yield pressure (P), above which MSCs will pass through microvessels of a given diameter. The objectives of the current study were to delineate the dependency of P on cell size and the heterogeneity of cell mechanical properties and diameters (D) of cultured MSCs.

Inhibition of inflammation induced shedding of the endothelial glycocalyx with low molecular weight heparin.

The endothelial surface layer (ESL) consists of the endothelial cell (EC) glycocalyx and adsorbed proteins, and forms a barrier between blood and the EC. Enzymatic shedding of the ESL in response to cytokines may expose receptors for leukocyte (WBC) adhesion and increase vascular permeability. Thus, intravital microscopy was used to explore stabilization of the ESL with low molecular weight heparin (LMWH) to mitigate structural changes with inflammation.

Role of matrix metalloproteases in the kinetics of leukocyte-endothelial adhesion in post-capillary venules.

Background: The endothelial glycocalyx serves as a barrier to leukocyte (WBC)-endothelium (EC) adhesion. Shedding of glycans, by matrix metalloproteases (MMPs) exposes EC integrin receptors to facilitate firm adhesion. However, the effect of shedding on the strength of the adhesive bond remains to be determined.

New insights into the microvascular mechanisms of drag reducing polymers: effect on the cell-free layer.

Drag-reducing polymers (DRPs) significantly increase blood flow, tissue perfusion, and tissue oxygenation in various animal models. In rectangular channel microfluidic systems, DRPs were found to significantly reduce the near-wall cell-free layer (CFL) as well as modify traffic of red blood cells (RBC) into microchannel branches. In the current study we further investigated the mechanism by which DRP enhances microvascular perfusion.

The effect of doxycycline on shedding of the glycocalyx due to reactive oxygen species.

The structure and composition of the endothelial cell (EC) glycocalyx reflect a balance of the biosynthesis of glycans and their shear dependent removal. Shedding of glycans from the EC surface has been shown to occur in response to reactive oxygen species (ROS) and inflammatory mediators. Using sub-antimicrobial doses of doxycycline, a broad spectrum matrix metalloprotease (MMP) inhibitor, inhibition of chemoattractant induced glycan shedding has suggested that MMPs may be a major effector of the loss of glycans.

Shear-dependent adhesion of leukocytes and lectins to the endothelium and concurrent changes in thickness of the glycocalyx of post-capillary venules in the low-flow state.

Objective: To elucidate shear-dependent effects of deformation of the endothelial glycocalyx on adhesion of circulating ligands in post-capillary venules, and delineate effect of MMPs.

Methods: Adhesion of WBCs and lectin-coated FLMs (0.1 μm diameter) to EC of post-capillary venules in mesentery was examined during acute reductions in shear rates (γ·, hemorrhagic hypotension).

Modulation of pre-capillary arteriolar pressure with drag-reducing polymers: a novel method for enhancing microvascular perfusion.

Objective: We have shown that drag-reducing polymers (DRP) enhance capillary perfusion during severe coronary stenosis and increase red blood cell velocity in capillaries, through uncertain mechanisms. We hypothesize that DRP decreases pressure loss from the aorta to the arteriolar compartment.

Methods: Intravital microscopy of the rat cremaster muscle and measurement of pressure in arterioles (diameters 20-132 μm) was performed in 24 rats.

Protease Activity and the Role of the Endothelial Glycocalyx in Inflammation.

A new paradigm for governance of leukocyte-endothelium (WBC-EC) adhesion during the inflammatory process is presented in which shedding of the endothelial glycocalyx exposes adhesion molecules on the EC surface, which promotes WBC-EC adhesion. It is postulated that the effector of this shedding is the activation of extracellular proteases, one of which may be a member of the matrix metalloproteinase (MMP) family of zinc dependent endopetidases. This model for the role of the glycocalyx as a barrier to WBC-EC adhesion includes the additional participation of normally active extracellular proteolytic enzymes, i.

The endothelial glycocalyx as a barrier to leukocyte adhesion and its mediation by extracellular proteases.

The endothelial cell (EC) surface is coated with a layer of polysaccharides linked to membrane-bound and trans-membrane proteoglycans that comprise the glycocalyx, which is augmented by adsorbed proteins derived from the blood stream. This surface layer has been shown to affect hemodynamics in small blood vessels of the microcirculation, the resistance to flow, and leukocyte (WBC) to EC adhesion. Parallel studies of WBC-EC adhesion in response to chemoattractants and cytokines, and shedding of constituents of the glycocalyx, have suggested a role for activation of extracellular proteases in mediating the dynamics of WBC adhesion in response to inflammatory and ischemic stimuli.

Shedding of the endothelial glycocalyx in arterioles, capillaries, and venules and its effect on capillary hemodynamics during inflammation.

The endothelial glycocalyx has been identified as a barrier to transvascular exchange of fluid, macromolecules, and leukocyte-endothelium [endothelial cell (EC)] adhesion during the inflammatory process. Shedding of glycans and structural changes of the glycocalyx have been shown to occur in response to several agonists. To elucidate the effects of glycan shedding on microvascular hemodynamics and capillary resistance to flow, glycan shedding in microvessels in mesentery (rat) was induced by superfusion with 10(-7) M fMLP.

Relative roles of doxycycline and cation chelation in endothelial glycan shedding and adhesion of leukocytes.

Leukocyte [white blood cell (WBC)] adhesion and shedding of glycans from the endothelium [endothelial cells (ECs)] in response to the chemoattractant f-Met-Leu-Phe (fMLP) has been shown to be attenuated by topical inhibition of matrix metalloproteases (MMPs) with doxycycline (Doxy). Since Doxy also chelates divalent cations, these responses were studied to elucidate the relative roles of cation chelation and MMP inhibition. WBC-EC adhesion, WBC rolling flux, and WBC rolling velocity were studied in postcapillary venules in the rat mesentery during superfusion with the cation chelator EDTA or Doxy.

Composition of the endothelial glycocalyx and its relation to its thickness and diffusion of small solutes.

The endothelial glycocalyx is well endowed with the glycosaminoglycans (GAGs) heparan sulfate, chondroitin sulfate and hyaluronan. The current studies aimed to assess the relative contributions of each of these GAGs to the thickness and permeability of the glycocalyx layer by direct enzymatic removal of each using micropipettes to infuse heparinase, chondroitinase and hyaluronidase into post-capillary venules of the intestinal mesentery of the rat. The relative losses of GAGs due to enzymatic removal were compared with stimulated shedding of glycans induced by superfusing the mesentery with 10(-)(7)M fMLP.

Inhibition of glycan shedding and leukocyte-endothelial adhesion in postcapillary venules by suppression of matrixmetalloprotease activity with doxycycline.

Objective: The aims of this study were to examine the role of matrixmetalloproteinases (MMPs) in causing shedding of glycan components of the endothelial glycocalyx and delineate the efficacy of doxycycline as an inhibitor of white blood cell-endothelial cell (WBC-EC) adhesion and glycan shedding in postcapillary venules.

Methods: WBC-EC adhesion in postcapillary venules of mesentery (rat) was examined in response to superfusion with the chemoattractant, f-Met-Leu-Phe (fMLP). Glycan shedding was delineated by using fluorescently labeled microspheres (FLMs; 0.

Measurement of solute transport in the endothelial glycocalyx using indicator dilution techniques.

A new method is presented to quantify changes in permeability of the endothelial glycocalyx to small solutes and fluid flow using techniques of indicator dilution. Following infusion of a bolus of fluorescent solutes (either FITC or FITC conjugated Dextran70) into the rat mesenteric circulation, its transient dispersion through post-capillary venules was recorded and analyzed offline. To represent dispersion of solute as a function of radial position in a microvessel, a virtual transit time (VTT) was calculated from the first moment of fluorescence intensity-time curves.

Microvascular rheology and hemodynamics.

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.

Effect of fibrinogen on leukocyte margination and adhesion in postcapillary venules.

Objective: To quantitatively evaluate the role of fibrinogen (Fb) as a determinant of leukocyte (WBC) margination in postcapillary venules in light of its ability to induce red blood cell (RBC) aggregation with reductions in shear rate (gamma) and increase adhesiveness of WBCs to endothelium (EC).

Methods: Red cell aggregation (RCA), WBC margination (flux at the EC), rolling velocity, and adhesion to the EC were measured in rat mesenteric postcapillary venules upon reducing gamma, prior to and following systemic infusion of Fb. Proximal occlusion of feeding microvessels with a blunted probe facilitated reductions in gamma from 600 to 50 s(-1).

Inflammation- and ischemia-induced shedding of venular glycocalyx.

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.

Role of glycocalyx in leukocyte-endothelial cell adhesion.

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.