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. 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.

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. Together, these carbohydrates form a layer that varies in glycan content along the length of post-capillary venules where WBC-EC adhesion occurs. Thickness and porosity of the glycocalyx can vary dramatically during the inflammatory response as observed by increased infiltration and diffusion of macromolecules within the layer following activation of the EC by cytokines and chemoattractants. In models of inflammation in the living animal, the shedding of glycans and diminished thickness of the glycocalyx rapidly occur to facilitate penetration by the WBCs and adhesion to the EC. The primary effectors of glycan shedding appear to be metalloproteases and heparanase released by the EC. Retardation of glycan shedding and WBC-EC adhesion has been demonstrated in vivo using MMP inhibitors and low-molecular-weight heparin (LMWH), where the latter competitively binds to heparanase liberated by the EC. Together, these agents may serve to stabilize the ESL and provide a useful strategy for treatment of inflammatory disorders.

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 (PY), above which MSCs will pass through microvessels of a given diameter. The objectives of the current study were to delineate the dependency of PY on cell size and the heterogeneity of cell mechanical properties and diameters (DCELL) of cultured MSCs. To this end the transient filtration test was employed to elucidate the mean filtration pressure (〈PY〉) for an ensemble of pores of a given size (DPORE) similar to in vivo microvessels. Cultured MSCs had a log-normal distribution of cell diameters (DCELL) with a mean of 15.8 ± 0.73 SD µm. MSC clearance from track-etched polycarbonate filters was studied for pore diameters of 7.3-15.4 µm. The pressure required to clear cells from filters with 30-85 × 103 pores rose exponentially with the ratio λ = DCELL/DPORE for 1.1 ≤ λ ≤ 2.2. The clearance of cells from each filter was characterized by a log-normal distribution in PY, with a mean filtration pressure of 0.02 ≤ ã€ˆPY〉 ≤ 6.7 cmH2O. For λ ≤ 1.56, the yield pressure (PY) was well represented by the cortical shell model of a cell with a viscous interior encapsulated by a shell under cortical tension τ0 = 0.99 ± 0.42 SD dyn/cm. For λ > 1.56, the 〈PY〉 characteristic of the cell population rose exponentially with λ. Analysis of the mean filtration pressure (〈PY〉) of each sample suggested that the larger diameter cells that skewed the distribution of DCELL contributed to about 20% of the mean filtration pressure. Further, if all cells had the same deformability (i.e., PY as a function of λ) as the average cell population, then ã€ˆPY〉 would have risen an order of magnitude above the average from fivefold at λ = 1.56 to 200-fold at λ = 2.1. Comparison of ã€ˆPY〉 to published microvascular pressures suggested that ã€ˆPY〉 may exceed microvessel pressure drops for λ exceeding 2.1, and rise 14-fold above capillary pressure drop at λ = 3 leading to 100% sequestration. However, due to the large variance of in vivo microvascular pressures entrapment of MSCs may be mitigated. Thus it is suggested that selecting fractions of the MSC population according to cell deformability may permit optimization of entrapment at sites targeted for tissue regeneration.

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. Following bolus infusions (i.v.) of LMWH (0.12-1.6mg/kg), shedding of glycans in response to 10-7M fMLP was measured by loss of fluorescently labeled lectins bound to the EC and WBC-EC adhesion was monitored in post-capillary venules of rat mesentery. During a 30min exposure to fMLP, a 50% reduction in fluorescence (indicative of glycan shedding) occurred at the lowest dose of LMWH whereas a 50% increase occurred (indicative of ESL compaction) at the highest dose. Shedding was reduced by LMWH in a dose dependent manner with an EC50 of 0.6mg/kg. Concomitant WBC-EC adhesion increased over 3-fold for all doses of LMWH. However, at a dose of 1.6mg/kg, WBC-EC adhesion did not rise significantly during the initial 10min exposure to fMLP. Correlation of WBC adhesion with intensity of the lectin stain for all measurements revealed a significant 40% reduction in adhesion as intensity increased 50%. This relationship was attributed to LMWH inhibition of heparanase and/or binding to components of the glycocalyx that resulted in mitigation of glycan shedding, compaction of the lectin stain and stabilization of the glycocalyx.

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. OBJECTIVES: Examine the effect of MMP inhibition on the kinetics of WBC-EC adhesion under normal and inflammatory conditions to delineate differences in the duration and number of adhesive bonds. METHODS: WBC adhesion in post-capillary venules was observed in rat mesentery. Adhesion duration and off-rates (KOFF) were correlated with shear stress during adhesion in response to 1 µM fMLP or 0.5 µM doxycycline (doxy, to inhibit MMP activation). RESULTS: Doxy increased mean adhesion time significantly from 2.5 (control) to 5.6 s, whereas fMLP increased it 8-fold to 20 s, which was not affected by pre-treatment with doxy. Estimates of the number of adhesive bonds (simplified Bell-model) revealed a significantly greater increase with fMLP compared to doxy alone, with no effect on fMLP by pretreatment with doxy. With doxy alone, KOFF was significantly 4-fold greater compared to fMLP, suggesting a much weaker bond. CONCLUSIONS: Although the increased number of bonds by MMP inhibition with doxy alone and fMLP were similar, the bonds due to doxy appeared weaker as evidenced by their shorter duration, and lesser reduction in KOFF relative to control. Thus doxy limits the availability of integrin binding sites during fMLP stimulated adhesion, but has a pro-adhesive effect due to increased ligands for WBC binding that arises from inhibition of normal sheddase activity on the EC.

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. We studied the effect of various concentrations of DRP on RBC distribution in more relevant round microchannels and the effect of DRP on CFL in the rat cremaster muscle in vivo. In round microchannels hematocrit was measured in parent and daughter branch at baseline and after addition of DRP. At DRP concentrations of 5 and 10 ppm, the plasma skimming effect in the daughter branch was eliminated, as parent and daughter branch hematocrit were equivalent, compared to a significantly lowered hematocrit in the daughter branch without DRPs. In anesthetized rats (N=11) CFL was measured in the cremaster muscle tissue in arterioles with a diameter of 32.6 ± 1.7 µm. In the control group (saline, N=6) there was a significant increase in CFL in time compared to corresponding baseline. Addition of DRP at 1 ppm (N=5) reduced CFL significantly compared to corresponding baseline and the control group. After DRP administration the CFL reduced to about 85% of baseline at 5, 15, 25 and 35 minutes after DRP infusion was complete. These in vivo and in vitro findings demonstrate that DRPs induce a reduction in CFL width and plasma skimming in the microvasculature. This may lead to an increase of RBC flux into the capillary bed, and thus explain previous observations of a DRP mediated enhancement of capillary 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. However, it has also been reported that doxycycline is a scavenger of ROS that may also activate MMPs. To clarify the basis for doxycycline as an inhibitor of glycan shedding, the present studies were undertaken to determine its effect on ROS induced shedding in post-capillary venules of the exteriorized mesentery of the rat. To this end, hypoxanthine (HX) and xanthine oxidase (XO) were rapidly mixed on the mesenteric surface for a 2min period to generate superoxide anion (O2(-)·) and the time course of glycan shedding was monitored in post-capillary venules over a 30min period. Glycan shedding was quantitated by loss of adherent fluorescently labeled lectin coated microspheres (FLMs, 0.1µm diameter) that were systemically infused. It was found that HX/XO caused FLM adhesion to decrease 45% within 30min. This effect could be inhibited in a dose dependent manner by the addition of superoxide dismutase to the superfusion solution, thus confirming the role of O2(-)·. In contrast, 0.5µM doxycycline had no effect on FLM shedding in response to HX/XO, contrary to its ability to attenuate shedding in response to the chemoattractant fMLP. Thus it is suggested that the efficacy of doxycycline as an inhibitor of glycan shedding during inflammation arises from its ability to inhibit MMP activation.

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). Adhesion was examined with or without superfusion with 0.5 µm doxycycline to inhibit MMPs. Thickness of the glycocalyx was measured by exclusion of fluorescent 70 kDa dextran from the EC surface. RESULTS: During superfusion with Ringers, rapid reductions in γ· resulted in a significant rise in WBC adhesion and a twofold rise in microsphere adhesion. With addition of doxycycline WBC and FLM adhesion increased twofold under high- and low-flow conditions. FLM adhesion was invariant with γ· throughout the network in the normal (high)-flow state. With reductions in γ·, thickness of the glycocalyx increased significantly, with or without doxycycline. CONCLUSIONS: The concurrent increase in WBC and FLM adhesion with increased thickness of the glycocalyx during reductions in shear suggests that glycocalyx core proteins recoil from their deformed steady-state configuration, which increases exposure of binding sites for circulating ligands.

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. DRP (polyethylene oxide, 1 ppm) was infused i.v. and measurements were made at baseline and 20 minutes after completion of DRP infusion. In a 10-rat subset, additional measurements were made three minutes after the start, and one to five and 10 minutes after completion of DRP. RESULTS: Twenty minutes after the completion of DRP, mean arteriolar pressure was 22% higher than baseline (from 42 ± 3 to 49 ± 3 mmHg, p < 0.005, n = 24). DRP decreased the pressure loss from the aorta to the arterioles by 24% (from 35 ± 6 to 27 ± 5 mmHg, p = 0.001, n = 10). In addition, there was a strong trend toward an increase in pressure at 10 minutes after the completion of DRP (n = 10). CONCLUSIONS: Drag-reducing polymers diminish pressure loss between the aorta and the arterioles. This results in a higher pre-capillary pressure and probably explains the observed DRP enhancement in capillary perfusion.

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. Likely candidates among the many proteases present are the matrix metalloproteases (MMPs). Inhibition of MMP activation with sub-antimicrobial doses of doxycycline, or zinc chelators, has also inhibited WBC adhesion and shedding of glycans from the EC surface in response to the chemoattractant fMLP. Taken together, these studies suggest that shedding of the EC glycocalyx exposes adhesion receptors and thus enhances WBC-EC adhesion. Future therapeutic strategies for treating pathologies such as the low flow state and inflammation may benefit by further exploration of the mechanics of the glycocalyx in light of protease activation and shear-dependent effects.

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.e. sheddases, which may cleave proteoglycans or activate lyases that cleave GAG chains in the glycocalyx. In support of this hypothesis, studies are examined which have established the concurrent activation of MMP proenzymes on the EC surface, shedding of the glycocalyx, and enhanced WBC-EC adhesion.

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. Shedding was quantified by reductions of fluorescently labeled lectin (BS-1) bound to the EC and reductions in thickness of the barrier to infiltration of 70-kDa dextran on the EC surface. Red cell velocities (two-slit technique), pressure drops (dual servo-null method), and capillary hematocrit (direct cell counting) were measured in parallel experiments. The results indicate that fMLP caused shedding of glycans in all microvessels with reductions in thickness of the barrier to 70-kDa dextran of 110, 80, and 123 nm, in arterioles, capillaries, and venules, respectively. Intravascular volumetric flows fell proportionately in all three divisions in response to rapid obstruction of venules by white blood cell (WBC)-EC adhesion, and capillary resistance to flow rose 18% due to diminished deformability of activated WBCs. Capillary resistance fell significantly 26% over a 30-min period, as glycans were shed from the EC surface to increase effective capillary diameter, whereas capillary hematocrit and anatomic diameter remained invariant. This decrease in capillary resistance mitigates the increase in resistance due to diminished WBC deformability, and hence these concurrent rheological events may be of equal importance in affecting capillary flow during the inflammatory process.

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. Shedding and accumulation of glycans on ECs, with and without fMLP, were quantified by the surface concentration of lectin (BS-1)-coated fluorescently labeled microspheres (FLMs) during constant circulating concentration. Without fMLP, low concentrations of EDTA (1-3 mM) increased FLM-EC sequestration due to disruption of the permeability barrier with prolonged exposure. In contrast, with 0.5 µM Doxy alone, FLM adhesion remained constant (i.e., no change in glycan content) on ECs, and WBC adhesion increased with prolonged superfusion. Without fMLP, EDTA did not affect firm WBC-EC adhesion but reduced WBC rolling flux in a dose-dependent manner. With fMLP, EDTA did not inhibit WBC adhesion, whereas Doxy did during the first 20 min of superfusion. Thus, the inhibition by Doxy of glycan (FLM) shedding and WBC adhesion in response to fMLP results from MMP inhibition, in contrast to cation chelation. With either Doxy or the MMP inhibitor GM-6001, WBC rolling velocity decreased by 50%, as in the case with fMLP, suggesting that MMP inhibition reduces sheddase activity, which increases the adhesiveness of rolling WBCs. These events increase the effective leukocrit on the venular wall and increase firm WBC-EC adhesion. Thus, MMP inhibitors have both a proadhesion effect by reducing sheddase activity while exerting an antiadhesion effect by inhibiting glycocalyx shedding and subsequent exposure of adhesion molecules on the EC surface.

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. Thickness of the glycocalyx was assessed by infiltration of the glycocalyx with circulating FITC labeled 70kDa dextran (Dx70) and measuring the distance from the dye front to the surface of the endothelium (EC), which averaged 463nm under control conditions. Reductions in thickness were 43.3%, 34.1% and 26.1% following heparinase, chondroitinase and hyaluronidase, respectively, and 89.7% with a mixture of all three enzymes. Diffusion coefficients of FITC in the glycocalyx were determined using a 1-D diffusion model. By comparison of measured transients in radial intensity of a bolus of FITC with that of a computational model a diffusion coefficient D was obtained. Values of D were obtained corresponding to the thickness of the layer demarcated by Dx70 (D(Dx70)), and a smaller sublayer 173nm above the EC surface (D(173)), prior to and following enzyme infusion and superfusion with fMLP. The magnitude of D(Dx70) was twice that of D(173) suggesting that the glycocalyx is more compact near the EC surface. Chondroitinase and hyaluronidase significantly increased both D(Dx70) and D(173). However, heparinase decreased D(Dx70), and did not induce any significant change for the D(173). These observations suggest that the three GAGs are not evenly distributed throughout the glycocalyx and that they each contribute to permeability of the glycocalyx to a differing extent. The fMLP-induced shedding caused a reduction in glycocalyx thickness (which may increase permeability) and as with heparinase, decreased the diffusion coefficient of solutes (which may decrease permeability). This behavior suggests that the removal of heparan sulfate may cause a collapse of the glycocalyx which counters decreases in thickness by compacting the layer to maintain a constant resistance to filtration.

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; 0.1 microm in diameter) coated with lectins and infused into the systemic circulation. The shedding of FLMs in response to fMLP was examined during superfusion with graded concentrations of doxycycline and the zinc chelator, ilomastat. RESULTS: Superfusion of mesentery with 10(-7) M of fMLP caused a reduction in FLM adhesion due to shedding of the glycocalyx and a rise in WBC-EC adhesion. WBC-EC adhesion and FLM shedding were reduced with subantimicrobial concentrations of doxycycline equal to or greater than 0.5 muM with an EC(50) value of 0.15 microM. MMP activation was verified by inhibition of shedding and attenuation of circulating MMP substrate cleavage at the venular wall with the zinc chelator, ilomastat (GM6001, 2.6 microM; US Biological, Swampscott, Massachusetts, USA). CONCLUSIONS: MMPs play a significant role in glycan shedding and WBC-EC adhesion, and doxycycline may stabilize the endothelial glycocalyx by inhibition of MMP activation.

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. Computer simulations and subsequent in vivo measurements showed that the radial gradient of VTT within the glycocalyx layer (Delta VTT/Delta r) may be related to the hydraulic resistance within the layer along the axial direction in a post-capillary venule and the effective diffusion coefficient within the glycocalyx. Modeling the inflammatory process by superfusion of the mesentery with 10(-7) M fMLP, Delta VTT/Delta r was found to decrease significantly from 0.23 +/- 0.08 SD s/microm to 0.18 +/- 0.09 SD s/microm. Computer simulations demonstrated that Delta VTT/Delta r is principally determined by three independent variables: glycocalyx thickness (delta), hydraulic resistivity (K(r)) and effective diffusion coefficient of the solute (D(eff)) within the glycocalyx. Based upon these simulations, the measured 20% decrease in Delta VTT/Delta r at the endothelial cell surface corresponds to a 20% increase in D(eff) over a broad range in K(r), assuming a constant thickness delta. The absolute magnitude of D(eff) required to match Delta VTT/Delta r between in vivo measurements and simulations was found to be on the order of 2.5 x 10(-3) x D(free), where D(free) is the diffusion coefficient of FITC in aqueous media. Thus the present method may provide a useful tool for elucidating structural and molecular alterations in the glycocalyx as occur with ischemia, metabolic and inflammatory events.