The mechanisms and physiological relevance of glycocalyx degradation in hepatic ischemia/reperfusion injury

Abstract

Significance: Hepatic ischemia/reperfusion (I/R) injury is an inevitable side effect of major liver surgery that can culminate in liver failure. The bulk of I/R-induced liver injury results from an overproduction of reactive oxygen and nitrogen species (ROS/RNS), which inflict both parenchymal and microcirculatory damage. A structure that is particularly prone to oxidative attack and modification is the glycocalyx (GCX), a meshwork of proteoglycans and glycosaminoglycans (GAGs) that covers the lumenal endothelial surface and safeguards microvascular homeostasis. ROS/RNS-mediated degradation of the GCX may exacerbate I/R injury by, for example, inducing vasoconstriction, facilitating leukocyte adherence, and directly activating innate immune cells.

Recent advances: Preliminary experiments revealed that hepatic sinusoids contain a functional GCX that is damaged during murine hepatic I/R and major liver surgery in patients. There are three ROS that mediate GCX degradation: hydroxyl radicals, carbonate radical anions, and hypochlorous acid (HOCl). HOCl converts GAGs in the GCX to GAG chloramides that become site-specific targets for oxidizing and reducing species and are more efficiently fragmented than the parent molecules. In addition to ROS/RNS, the GAG-degrading enzyme heparanase acts at the endothelial surface to shed the GCX.

Critical issues: The GCX seems to be degraded during major liver surgery, but the underlying cause remains ill-defined.

Future directions: The relative contribution of the different ROS and RNS intermediates to GCX degradation in vivo, the immunogenic potential of the shed GCX fragments, and the role of heparanase in liver I/R injury all warrant further investigation.

FIG. 1.

GCX structure. The backbone of the GCX is composed of syndecan and glypican PGs that are anchored in the endothelial cell membrane. Long sulfated GAG (HS [in green] and CS [in orange]) side chains are covalently attached to the PG cores. The nonsulfated GAG HA (in red) is not bound to a PG but attaches to the GCX by directly associating with CS chains or endothelial surface receptors such as CD44 (not shown). CS, chondroitin sulfate; GAG, glycosaminoglycan; GCX, glycocalyx; HA, hyaluronic acid; HS, heparan sulfate; PG, proteoglycan. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 2.

GAG structure. GAGs are attached to the proteoglycan core via a tetrasaccharide linkage composed of Xyl, Gal (2×), and GluA. Next, HS or CS are produced by respectively adding GlcNAc or GalNAc to the GluA of the tetrasaccharide linker. X and Y indicate variable substitution of amino- or O-groups, respectively (see in-figure legend). Gal, galactose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; GluA, glucuronic acid; Xyl, xylose. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 3.

Schematic representation of GCX degradation during hepatic I/R injury. An intact GCX (shown on the left) maintains vascular homeostasis by, for example, preventing leukocyte adherence (see the GCX Function section). During reperfusion, the production of ROS/RNS (green) by SECs and leukocytes (e.g., monocytes, neutrophils) and the release of heparanase by SECs (yellow) could lead to GCX degradation. The loss of GCX not only abrogates the protective functions of the GCX but additionally activates the immune system (shown on the right). Circulating GCX fragments can be detected by immune receptors on the surface of Kupffer cells and SECs, thereby inciting the production of proinflammatory mediators such as TNF-α (see the Proinflammatory Consequences of GCX Degradation section). I/R, ischemia/reperfusion; RNS, reactive nitrogen species; ROS, reactive oxygen species; SEC, sinusoidal endothelial cell; TNF-α, tumor necrosis factor alpha. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 4.

Intravital two-photon microscopy of the hepatic GCX in mice. (A–C) Show the hepatic sinusoids as visualized by intravital two-photon microscopy in male C57Bl/6 mice (N=2) following the intravenous infusion of a neutral 40-kDa Texas Red-dextran [(A), red fluorescence] and an anionic 150-kDa FITC-dextran [(B), green fluorescence]. The overlay of (A) and (B) is shown in (C). The fluorescent column diameters are shown in the insets that correspond to the region delineated by the dashed marquee. Inasmuch as the smaller neutral (red) dye penetrates into the GCX, whereas the larger and anionic (green) dye is excluded by the GCX (159), the width of the GCX per vessel can be measured by subtracting the diameter of the FITC (green) fluorescence column from the diameter of the Texas Red (red) fluorescence column and dividing this number by 2. The inset in (A) shows a representative measurement of a Texas Red fluorescence column, which is superimposed on the FITC fluorescence column at the exact same position in (B) to give the composite image in (C), revealing a diameter difference that is most likely attributable to FITC-dextran exclusion by the GCX. This is further illustrated in the overlay (C), where it appears that the hepatic sinusoids are lined by exclusively red fluorescence (arrowheads), again indicating that the green dye does not penetrate to the vascular wall due to spatial exclusion by the GCX. (D) Shows the calculated GCX width for 44 regions of interest, yielding a median GCX size of 0.193 μm (interquartile range=0.329 μm). Error bars (shown in red) also represent the median and interquartile range. The results were validated by an observer that was blinded to the experimental design (Supplementary Fig. S2). (E–G) Indicate that the width difference between the two fluorescence columns seems absent in post-sinusoidal venules, implying that the GCX may only be present in the sinusoids. The intravital imaging procedure is described in detail in the Supplementary Data. FITC, fluorescein isothiocyanate. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 5.

The formation of ROS and RNS during hepatic I/R injury. The combined presence of the template radicals •NO and O₂^•− ultimately gives rise to three species, shown in red, that directly induce GCX modification or fragmentation: CO₃^•−, •OH, and HOCl. The enzymatic and metal catalysts are indicated in green, see the Mechanisms of GCX Oxidation section for details. •OH, hydroxyl radical; •NO, nitric oxide; CO₃^•−, carbonate radical anion; eNOS, (uncoupled) endothelial nitric oxide synthase; HOCl, hypochlorous acid; iNOS, inducible nitric oxide synthase; MPO, myeloperoxidase; NOX2, phagocyte NADPH oxidase; O₂^•−, superoxide anion; SOD, superoxide dismutase; TM, transition metal. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 6.

Possible molecular fates of nitrogen-centered (amidyl) radicals that are formed upon oxidation or reduction of GAG chloramides. The nitrogen-centered radicals are short-lived and rearrange via intramolecular proton transfer to either a C-2 radical on the GlcNAc moiety (left pathway) or C-4 radicals on the GluA moiety (right pathway). Both radicals can rearrange via nonfragmentation pathways or induce GAG strand cleavage. The relative importance of each pathway is detailed in the Modification and Fragmentation of GAGs by Nonradical Halogen-Containing Oxidants section. The fate of the C-2 and C-4 radicals is also discussed in Refs. (117, 118, 132, 133). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 7.

Proposed mechanism for the reaction of O₂^•− with GAG chloramides. Reduction of the chloramide initially yields a nitrogen-centered (amidyl) radical (top right) that subsequently reacts with O₂ to form a peroxy radical intermediate (N−O−O^•, left center). Two GAG peroxy radicals subsequently react to ultimately form two stable GAG nitroxides (bottom) without inducing GAG chain scission. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 8.

HS is released into the circulation following hepatic I/R in mice. Male C57Bl/6J mice (8–12 weeks old) were subjected to 60 min of partial liver ischemia or a sham operation (N=5–6/group). Ethylenediaminetetraaceticacid-antiocoagulated blood samples were collected after 1 or 6 h of reperfusion or 6 h after sham operation. Plasma HS levels were assessed using the General Heparan Sulfate ELISA kit from Amsbio (catalog no. E0623Ge; Milton, United Kingdom). Data are shown as mean±standard deviation and *indicates p<0.025 compared to the sham group. Statistical analysis was performed using the Kruskal–Wallis test and post hoc Mann–Whitney U tests with an adjusted significance level of 0.025 to correct for multiple testing. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 9.

Circulating HS levels in patients who had undergone a major liver resection. Serum samples were obtained from all patients (N=18) before liver resection and after 1 and 6 h of reperfusion (I/R group) or 1 and 6 h after resection (CTRL group). Patient characteristics are included in Supplementary Table S1. Serum HS content was assessed by enzyme-linked immunosorbent assay (Fig. 6) and is reported as mean±SEM. The results were normalized to plasma protein content, determined with the Pierce Total Protein Assay Kit (Rockford, IL), to correct for hemodilution. The red squares indicate the mean per time point. Intragroup differences were assessed using the Friedman test for repeated measurements in combination with Wilcoxon signed-rank post hoc testing (t=1 and t=6 vs. t=0). Because the significance level was adjusted to correct for multiple testing, * and ^# indicate p<0.025 compared to t=0 in the CTRL and I/R group, respectively. In the CTRL group (A), HS concentrations were 0.98±0.48 ng/mg protein at baseline, increased to 2.13±0.86 ng/mg protein 1 h after liver resection, and decreased to 0.43±0.15 ng/mg protein 6 h after liver resection. In the I/R group (C), the HS concentration was 1.85±0.79 ng/mg protein at baseline, increased to 2.88±1.09 ng/mg protein after 1 h of reperfusion, and dropped to 0.30±0.078 ng/mg protein after 6 h of reperfusion. The same data are plotted as fold increase compared to baseline in (B, D, E), yielding a peak mean fold increase of 3.55±0.94 1 h after liver resection in the CTRL group and a mean±SEM peak fold increase of 5.28±2.96 after 1 h of reperfusion in the I/R group. (F, G) Show correlation plots and linear regression lines of the cumulative duration of ischemia plotted against the peak in postoperative ALT [a liver damage marker, (F)] and postoperative peak fold increase in circulating HS (G). (H) Shows the correlation between the postoperative peak ALT and peak HS fold increase. ALT, alanine aminotransferase; CTRL, control; SEM, standard error of the mean. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars