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.

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.

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.