Doxycycline protects against sepsis-induced endothelial glycocalyx shedding.

Endothelial glycocalyx (eGC) covers the inner surface of the vessels and plays a role in vascular homeostasis. Syndecan is considered the "backbone" of this structure. Several studies have shown eGC shedding in sepsis and its involvement in organ dysfunction. Matrix metalloproteinases (MMP) contribute to eGC shedding through their ability for syndecan-1 cleavage. This study aimed to investigate if doxycycline, a potent MMP inhibitor, could protect against eGC shedding in lipopolysaccharide (LPS)-induced sepsis and if it could interrupt the vascular hyperpermeability, neutrophil transmigration, and microvascular impairment. Rats that received pretreatment with doxycycline before LPS displayed ultrastructural preservation of the eGC observed using transmission electronic microscopy of the lung and heart. In addition, these animals exhibited lower serum syndecan-1 levels, a biomarker of eGC injury, and lower perfused boundary region (PBR) in the mesenteric video capillaroscopy, which is inversely related to the eGC thickness compared with rats that only received LPS. Furthermore, this study revealed that doxycycline decreased sepsis-related vascular hyperpermeability in the lung and heart, reduced neutrophil transmigration in the peritoneal lavage and inside the lungs, and improved some microvascular parameters. These findings suggest that doxycycline protects against LPS-induced eGC shedding, and it could reduce vascular hyperpermeability, neutrophils transmigration, and microvascular impairment.

EFFECT OF MIR-21-3P ON INTESTINAL INJURY IN RATS WITH TRAUMATIC HEMORRHAGIC SHOCK RESUSCITATED WITH THE SODIUM BICARBONATE RINGER'S SOLUTION.

ABSTRACT: Background : This study aims to determine the impact and mechanism of miR-21-3p on intestinal injury and intestinal glycocalyx during fluid resuscitation in traumatic hemorrhagic shock (THS), and the different impacts of sodium lactate Ringer's solution (LRS) and sodium bicarbonate Ringer's solution (BRS) for resuscitation on intestinal damage. Methods : A rat model of THS was induced by hemorrhage from the left femur fracture. The pathological changes of intestinal tissues and glycocalyx structure were observed by hematoxylin-eosin staining and transmission electron microscope. MiR-21-3p expression in intestinal tissues was detected by real-time quantitative polymerase chain reaction. The expression of glycocalyx-, cell junction-, and PI3K/Akt/NF-κB signaling pathway-related proteins was analyzed by western blot. Results : MiR-21-3p expression was increased in THS rats, which was suppressed by resuscitation with BRS. BRS or LRS aggravated the intestinal injury and damaged intestinal glycocalyx in THS rats. The expression of SDC-1, HPA, ß-catenin, MMP2, and MMP9 was upregulated, the expression of E-cad was downregulated, and the PI3K/Akt/NF-κB signaling pathway was activated in THS rats, which were further aggravated by BRS or LRS. The adverse effect of LRS was more serious than BRS. MiR-21-3p overexpression deteriorated the injury of intestinal tissues and intestinal glycocalyx; increased the expression of SDC-1, HPA, ß-catenin, MMP2, and MMP9 while decreasing E-cad expression; and activated the PI3K/Akt/NF-κB signaling pathway in BRS-resuscitated THS rats. Conclusion : MiR-21-3p aggravated intestinal tissue injury and intestinal glycocalyx damage through activating PI3K/Akt/NF-κB signaling pathway in rats with THS resuscitated with BRS.

Adiponectin Reduces Glomerular Endothelial Glycocalyx Disruption and Restores Glomerular Barrier Function in a Mouse Model of Type 2 Diabetes.

Adiponectin has vascular anti-inflammatory and protective effects. Although adiponectin protects against the development of albuminuria, historically, the focus has been on podocyte protection within the glomerular filtration barrier (GFB). The first barrier to albumin in the GFB is the endothelial glycocalyx (eGlx), a surface gel-like barrier covering glomerular endothelial cells (GEnCs). In diabetes, eGlx dysfunction occurs before podocyte damage; hence, we hypothesized that adiponectin could protect from eGlx damage to prevent early vascular damage in diabetic kidney disease (DKD). Globular adiponectin (gAd) activated AMPK signaling in human GEnCs through AdipoR1. It significantly reduced eGlx shedding and the tumor necrosis factor-α (TNF-α)-mediated increase in syndecan-4 (SDC4) and MMP2 mRNA expression in GEnCs in vitro. It protected against increased TNF-α mRNA expression in glomeruli isolated from db/db mice and against expression of genes associated with glycocalyx shedding (namely, SDC4, MMP2, and MMP9). In addition, gAd protected against increased glomerular albumin permeability (Ps'alb) in glomeruli isolated from db/db mice when administered intraperitoneally and when applied directly to glomeruli (ex vivo). Ps'alb was inversely correlated with eGlx depth in vivo. In summary, adiponectin restored eGlx depth, which was correlated with improved glomerular barrier function, in diabetes.

Low dose cadmium inhibits syndecan-4 expression in glycocalyx of glomerular endothelial cells.

Cadmium (Cd) is one of the most polluting heavy metal in the environment. Cd exposure has been elucidated to cause dysfunction of the glomerular filtration barrier (GFB). However, the underlying mechanism remains unclear. C57BL/6J male mice were administered with 2.28 mg/kg cadmium chloride (CdCl2) dissolved in distilled water by oral gavage for 14 days. The expression of SDC4 in the kidney tissues was detected. Human renal glomerular endothelial cells (HRGECs) were exposed to varying concentrations of CdCl2 for 24 h. The mRNA levels of SDC4, along with matrix metalloproteinase (MMP)-2 and 9, were analyzed by quantitative PCR. Additionally, the protein expression levels of SDC4, MMP-2/9, and both total and phosphorylated forms of Smad2/3 (P-Smad2/3) were detected by western blot. The extravasation rate of fluorescein isothiocyanate-dextran through the Transwell was used to evaluate the permeability of HRGECs. SB431542 was used as an inhibitor of transforming growth factor (TGF)-ß signaling pathway to further investigate the role of TGF-ß. Cd reduced SDC4 expression in both mouse kidney tissues and HRGECs. In addition, Cd exposure increased permeability and upregulated P-Smad2/3 levels in HRGECs. SB431542 treatment inhibited the phosphorylation of Smad2/3, Cd-induced SDC4 downregulation, and hyperpermeability. MMP-2/9 levels increased by Cd exposure was also blocked by SB431542, demonstrating the involvement of TGF-ß/Smad pathway in low-dose Cd-induced SDC4 reduction in HRGECs. Given that SDC4 is an essential component of glycocalyx, protection or repair of endothelial glycocalyx is a potential strategy for preventing or treating kidney diseases associated with environmental Cd exposure.

Endothelial Glycocalyx Integrity in Treatment-Naïve People Living with HIV before and One Year after Antiretroviral Treatment Initiation.

Endothelial glycocalyx (EG) derangement has been associated with cardiovascular disease (CVD). Studies on EG integrity among people living with HIV (PLWH), are lacking. We conducted a prospective cohort study among treatment-naïve PLWH who received emtricitabine/tenofovir alafenamide, combined with either an integrase strand transfer inhibitor (INSTI, dolutegravir, raltegravir or elvitegravir/cobicistat), or a protease inhibitor (PI, darunavir/cobicistat). We assessed EG at baseline, 24 (±4) and 48 (±4) weeks, by measuring the perfused boundary region (PBR, inversely proportional to EG thickness), in sublingual microvessels. In total, 66 consecutive PLWH (60 (90.9%) males) with a median age (interquartile range, IQR) of 37 (12) years, were enrolled. In total, 40(60.6%) received INSTI-based regimens. The mean (standard deviation) PBR decreased significantly from 2.17 (0.29) µm at baseline to 2.04 (0.26) µm (p = 0.019), and then to 1.93 (0.3) µm (p < 0.0001) at 24 (±4) and 48 (±4) weeks, respectively. PBR did not differ among treatment groups. PLWH on INSTIs had a significant PBR reduction at 48 (±4) weeks. Smokers and PLWH with low levels of viremia experienced the greatest PBR reduction. This study is the first to report the benefit of antiretroviral treatment on EG improvement in treatment-naïve PLWH and depicts a potential bedside biomarker and therapeutic target for CVD in PLWH.

Mineralocorticoid receptor-antagonism prevents COVID-19-dependent glycocalyx damage.

Proinflammatory cytokines target vascular endothelial cells during COVID-19 infections. In particular, the endothelial glycocalyx (eGC), a proteoglycan-rich layer on top of endothelial cells, was identified as a vulnerable, vasoprotective structure during infections. Thus, eGC damage can be seen as a hallmark in the development of endothelial dysfunction and inflammatory processes. Using sera derived from patients suffering from COVID-19, we could demonstrate that the eGC became progressively worse in relation to disease severity (mild vs severe course) and in correlation to IL-6 levels. This could be prevented by administering low doses of spironolactone, a well-known and highly specific aldosterone receptor antagonist. Our results confirm that SARS-CoV-2 infections cause eGC damage and endothelial dysfunction and we outline the underlying mechanisms and suggest potential therapeutic options.

Anti-Inflammatory Activity of Orally Administered Monostroma nitidum Rhamnan Sulfate against Lipopolysaccharide-Induced Damage to Mouse Organs and Vascular Endothelium.

We previously reported that rhamnan sulfate (RS) purified from Monostroma nitidum significantly suppressed lipopolysaccharide (LPS)-induced inflammation in cultured human vascular endothelial cells. Here, we analyzed the effect of orally administered RS on LPS-induced damage to mouse organs and vascular endothelium. RS (1 mg) was orally administered daily to BALB/c mice, 50 µg of LPS was intraperitoneally administered on day 8, and Evans blue was injected into the tail vein 6 h later. After 30 min, LPS-treated mice showed pulmonary Evans blue leakage and elevated plasma levels of liver damage markers, whereas this reaction was suppressed in LPS + RS-treated mice. Immunohistochemical and Western blot analysis of mouse organs 24 h after LPS treatment showed significant neutrophil infiltration into the lung, liver, and jejunum tissues of LPS-treated mice and high expression levels of inflammation-related factors in these tissues. Expression levels of these factors were significantly suppressed in LPS + RS-treated mice. Analysis of lung glycocalyx showed a significant reduction in glycocalyx in LPS-treated mice but not in LPS + RS-treated mice. Levels of syndecan-4, one of the glycocalyx components, decreased in LPS-treated mice and increased in LPS + RS-treated mice. The current results suggest that orally administered RS protects organs and vascular endothelium from LPS-induced inflammation and maintains blood circulation.

Plasma components to protect the endothelial barrier after shock: A role for sphingosine 1-phosphate.

BACKGROUND: Hemorrhagic shock leads to endothelial glycocalyx shedding, endothelial cellular inflammation, and increased vascular permeability. Early plasma administration improves survival in severely injured patients; this may be due in part to its ability to ameliorate this trauma-induced endotheliopathy. The protective effect of early plasma administration may be due to its sphingosine 1-phosphate content. Principle carriers of plasma sphingosine 1-phosphate include apolipoprotein M and albumin. The relative roles of these carriers on sphingosine 1-phosphate protective effects are unknown and were studied in an in vitro model of microcirculation. METHODS: Endothelial cell monolayers were established in microfluidic perfusion devices and exposed to control or biomimetic shock conditions. Sphingosine 1-phosphate, albumin + sphingosine 1-phosphate, or apolipoprotein M + sphingosine 1-phosphate were added later to the perfusate. Biomarkers of endothelial and glycocalyx activation and damage were then determined. RESULTS: Sphingosine 1-phosphate preserved endothelial and glycocalyx barrier function after exposure to conditions of shock in the microcirculation. The protective effect was related to sphingosine 1-phosphate chaperones; the apolipoprotein M loaded with sphingosine 1-phosphate had the most profound effect. CONCLUSION: Carrier-based sphingosine 1-phosphate may be a useful adjunct in early hemorrhagic shock resuscitation.

Endothelial Dysfunction in Fabry Disease Is Related to Glycocalyx Degradation.

Fabry disease (FD) is an X-linked multisystemic lysosomal storage disease due to a deficiency of α-galactosidase A (GLA/AGAL). Progressive cellular accumulation of the AGAL substrate globotriaosylceramide (Gb3) leads to endothelial dysfunction. Here, we analyzed endothelial function in vivo and in vitro in an AGAL-deficient genetic background to identify the processes underlying this small vessel disease. Arterial stiffness and endothelial function was prospectively measured in five males carrying GLA variants (control) and 22 FD patients under therapy. AGAL-deficient endothelial cells (EA.hy926) and monocytes (THP1) were used to analyze endothelial glycocalyx structure, function, and underlying inflammatory signals. Glycocalyx thickness and small vessel function improved significantly over time (p<0.05) in patients treated with enzyme replacement therapy (ERT, n=16) and chaperones (n=6). AGAL-deficient endothelial cells showed reduced glycocalyx and increased monocyte adhesion (p<0.05). In addition, increased expression of angiopoietin-2, heparanase and NF-κB was detected (all p<0.05). Incubation of wild-type endothelial cells with pathological globotriaosylsphingosine concentrations resulted in comparable findings. Treatment of AGAL-deficient cells with recombinant AGAL (p<0.01), heparin (p<0.01), anti-inflammatory (p<0.001) and antioxidant drugs (p<0.05), and a specific inhibitor (razuprotafib) of angiopoietin-1 receptor (Tie2) (p<0.05) improved glycocalyx structure and endothelial function in vitro. We conclude that chronic inflammation, including the release of heparanases, appears to be responsible for the degradation of the endothelial glycocalyx and may explain the endothelial dysfunction in FD. This process is partially reversible by FD-specific and anti-inflammatory treatment, such as targeted protective Tie2 treatment.

Intramuscular Exposure to a Lethal Dose of Ricin Toxin Leads to Endothelial Glycocalyx Shedding and Microvascular Flow Abnormality in Mice and Swine.

Ricin toxin isolated from the castor bean (Ricinus communis) is one of the most potent and lethal molecules known. While the pathophysiology and clinical consequences of ricin poisoning by the parenteral route, i.e., intramuscular penetration, have been described recently in various animal models, the preceding mechanism underlying the clinical manifestations of systemic ricin poisoning has not been completely defined. Here, we show that following intramuscular administration, ricin bound preferentially to the vasculature in both mice and swine, leading to coagulopathy and widespread hemorrhages. Increased levels of circulating VEGF and decreased expression of vascular VE-cadherin caused blood vessel impairment, thereby promoting hyperpermeability in various organs. Elevated levels of soluble heparan sulfate, hyaluronic acid and syndecan-1 were measured in blood samples following ricin intoxication, indicating that the vascular glycocalyx of both mice and swine underwent extensive damage. Finally, by using side-stream dark field intravital microscopy imaging, we determined that ricin poisoning leads to microvasculature malfunctioning, as manifested by aberrant blood flow and a significant decrease in the number of diffused microvessels. These findings, which suggest that glycocalyx shedding and microcirculation dysfunction play a major role in the pathology of systemic ricin poisoning, may serve for the formulation of specifically tailored therapies for treating parenteral ricin intoxication.

N-Glycomic Analysis of the Cell Shows Specific Effects of Glycosyl Transferase Inhibitors.

Glycomic profiling methods were used to determine the effect of metabolic inhibitors on glycan production. These inhibitors are commonly used to alter the cell surface glycosylation. However, structural analysis of the released glycans has been limited. In this research, the cell membranes were enriched and the glycans were released to obtain the N-glycans of the glycocalyx. Glycomic analysis using liquid chromatography-mass spectrometry (LC-MS) with a PGC chip column was used to profile the structures in the cell membrane. Glycans of untreated cells were compared to glycans of cells treated with inhibitors, including kifunensine, which inhibits the formation of complex- and hybrid-type structures, 2,4,7,8,9-Penta-O-acetyl-N-acetyl-3-fluoro-b-d-neuraminic acid methyl ester for sialylated glycans, 2-deoxy-2-fluorofucose, and 6-alkynyl fucose for fucosylated glycans. Kifunensine was the most effective, converting nearly 95% of glycans to high mannose types. The compound 6-alkynyl fucose inhibited some fucosylation but also incorporated into the glycan structure. Proteomic analysis of the enriched membrane for the four inhibitors showed only small changes in the proteome accompanied by large changes in the N-glycome for Caco-2. Future works may use these inhibitors to study the cellular behavior associated with the alteration of glycosylation in various biological systems, e.g., viral and bacterial infection, drug binding, and cell-cell interactions.

Inhalation of 2% Hydrogen Improves Survival Rate and Attenuates Shedding of Vascular Endothelial Glycocalyx in Rats with Heat Stroke.

ABSTRACT: Heat stroke is characterized by excessive oxidative stress and inflammatory responses, both of which are implicated in vascular endothelial glycocalyx shedding and heat-stroke mortality. Although molecular hydrogen has antioxidation and anti-inflammatory potency, its effect on the vascular endothelial glycocalyx in heat stroke has not been examined. Therefore, the aim of this study was to investigate the influence of hydrogen inhalation on the survival and thickness of the vascular endothelial glycocalyx of rats subjected to heat stroke. Altogether, 98 Wistar rats were assigned to the experiments. A heat-controlled chamber set at 40°C temperature and 60% humidity was used to induce heat stroke. After preparation, the anesthetized rats that underwent the heating process were subjected to an hour of stabilization in which 0%, 2%, or 4% hydrogen gas was inhaled and maintained until the experiment ended. In addition to survival rate assessments, blood samples and left ventricles were collected to evaluate the thickness of the vascular endothelial glycocalyx and relevant biomarkers. The results showed that 2% hydrogen gas significantly improved survival in the heat-stroked rats and partially preserved the thickness of the endothelial glycocalyx. In addition, serum levels of endotoxin, syndecan-1, malondialdehyde, and tumor necrosis factor-α decreased, whereas superoxide dismutase levels increased, indicating that inhalation of 2% hydrogen attenuated the damage to the vascular endothelial glycocalyx through its antioxidative and anti-inflammatory effects.

Roles of glomerular endothelial hyaluronan in the development of proteinuria.

Vascular endothelial cells are covered with glycocalyx comprising heparan sulfate, hyaluronan, chondroitin sulfate, and associated proteins. Glomerular endothelial glycocalyx is involved in protecting against induction of proteinuria and structural damage, but the specific components in glycocalyx that represent therapeutic targets remain unclear. Anti-vascular endothelial growth factor (VEGF) therapy is associated with an increased risk of glomerular endothelial injury. This study investigated whether hyaluronan could provide a therapeutic target to protect against proteinuria. We conducted ex vivo and in vivo experiments to explore the effects of degrading glomerular hyaluronan by administering hyaluronidase and of supplementation with hyaluronan. We investigated hyaluronan expression using biotin-labeled hyaluronan-binding protein (HABP) in human kidney specimens or serum hyaluronan in endothelial injuries under inhibition of VEGF signaling. We directly demonstrated hyaluronan in glomerular endothelial layers using HABP staining. Ex vivo and in vivo experiments showed the development of proteinuria after digestion of hyaluronan in glomerular capillaries. Supplementation with hyaluronan after hyaluronidase treatment suppressed proteinuria. Mice in the in vivo study developed albuminuria after intraperitoneal injection of hyaluronidase with decreased glomerular hyaluronan and increased serum hyaluronan. In human kidneys with endothelial cell dysfunction and proteinuria due to inhibition of VEGF, glomerular expression of hyaluronan was reduced even in normal-appearing glomeruli. Serum hyaluronan levels were elevated in patients with pre-eclampsia with VEGF signaling inhibition. Our data suggest that hyaluronan itself plays crucial roles in preventing proteinuria and preserving the integrity of endothelial cells. Hyaluronan could provide a therapeutic target for preventing glomerular endothelial glycocalyx damage, including VEGF signaling inhibition.

A Randomized, Multicenter, Open-Label, Blinded End Point, Phase 2, Feasibility, Efficacy, and Safety Trial of Preoperative Microvascular Protection in Patients Undergoing Major Abdominal Surgery.

BACKGROUND: The endothelial glycocalyx, a carbohydrate-rich layer coating all endothelial surfaces, plays a fundamental role in the function of microcirculation. The primary aim of this study was to evaluate the feasibility of using dexamethasone and albumin to protect the endothelial glycocalyx in patients undergoing abdominal surgery. Secondary and exploratory outcomes included efficacy and safety. METHODS: We conducted a multicenter, open-label, blinded end point, phase 2, randomized trial. Patients undergoing colorectal, pancreas, or liver surgery were recruited and randomized to receive either intravenous dexamethasone (16 mg) and 20% albumin (100 mL) at induction of anesthesia, then 200 mL of 20% albumin with each subsequent 1000 mL of crystalloid administered (dexamethasone and albumin [Dex-Alb] group), or crystalloid fluid only with no dexamethasone (control group). Feasibility end points included patient recruitment and retention, consent rate, and successful study drug administration. The primary efficacy end point was the measurement of plasma syndecan-1 level on postoperative day (POD) 1, and secondary end points were heparan sulfate levels and inflammatory markers measured at 4 perioperative timepoints. Safety end points included errors in administration of the intervention, hyperglycemia, occurrence of postoperative complications, and patient retention. RESULTS: Seventy-two patients were randomized. All feasibility end points were achievable. There were no statistically significant differences observed in median (interquartile range) syndecan-1 levels on POD 1 (39 ng·mL-1 [20-97] in the Dex-Alb group versus 41 ng·mL-1 [19-84] in the control group; difference in medians -2.1, 95% confidence interval [CI], -13 to 8.6; P = .69). The Dex-Alb group had lower POD 1 heparan sulfate levels (319 ng·mL-1 [161-717] in the Dex-Alb group versus 1422 [670-2430] ng·mL-1 in the control group; difference in medians -1085, 95% CI, -1779 to -391) and C-reactive protein (CRP) levels on POD 1 (48 [29-77] mg·L-1 in the Dex-Alb group versus 85 mg·L-1 [49-133] in the control group; difference in medians -48, 95% CI, -75 to -21). Fewer patients had one or more postoperative complication in the Dex-Alb group than in the control group (6 [17%] vs 18 patients [50%]; odds ratio = 0.2, 95% CI, 0.06-0.6). CONCLUSIONS: Intravenous dexamethasone and albumin administration was feasible but did not reduce syndecan-1 on POD 1 in patients undergoing abdominal surgery. Given the clinically important CIs observed between the groups for heparan sulfate, CRP, and postoperative complications, a larger trial assessing the associations between dexamethasone and albumin administration and these outcomes is warranted.

Protectin conjugates in tissue regeneration 1 restores lipopolysaccharide-induced pulmonary endothelial glycocalyx loss via ALX/SIRT1/NF-kappa B axis.

BACKGROUND: Endothelial glycocalyx loss is integral to increased pulmonary vascular permeability in sepsis-related acute lung injury. Protectin conjugates in tissue regeneration 1 (PCTR1) is a novel macrophage-derived lipid mediator exhibiting potential anti-inflammatory and pro-resolving benefits. METHODS: PCTR1 was administrated intraperitoneally with 100 ng/mouse after lipopolysaccharide (LPS) challenged. Survival rate and lung function were used to evaluate the protective effects of PCTR1. Lung inflammation response was observed by morphology and inflammatory cytokines level. Endothelial glycocalyx and its related key enzymes were measured by immunofluorescence, ELISA, and Western blot. Afterward, related-pathways inhibitors were used to identify the mechanism of endothelial glycocalyx response to PCTR1 in mice and human umbilical vein endothelial cells (HUVECs) after LPS administration. RESULTS: In vivo, we show that PCTR1 protects mice against lipopolysaccharide (LPS)-induced sepsis, as shown by enhanced the survival and pulmonary function, decreased the inflammatory response in lungs and peripheral levels of inflammatory cytokines such as tumor necrosis factor-α, interleukin-6, and interleukin-1ß. Moreover, PCTR1 restored lung vascular glycocalyx and reduced serum heparin sulphate (HS), syndecan-1 (SDC-1), and hyaluronic acid (HA) levels. Furthermore, we found that PCTR1 downregulated heparanase (HPA) expression to inhibit glycocalyx degradation and upregulated exostosin-1 (EXT-1) protein expression to promote glycocalyx reconstitution. Besides, we observed that BAY11-7082 blocked glycocalyx loss induced by LPS in vivo and in vitro, and BOC-2 (ALX antagonist) or EX527 (SIRT1 inhibitor) abolished the restoration of HS in response to PCTR1. CONCLUSION: PCTR1 protects endothelial glycocalyx via ALX receptor by regulating SIRT1/NF-κB pathway, suggesting PCTR1 may be a significant therapeutic target for sepsis-related acute lung injury.

Recombinant Antithrombin Attenuates Acute Respiratory Distress Syndrome in Experimental Endotoxemia.

Sepsis-induced endothelial acute respiratory distress syndrome is related to microvascular endothelial dysfunction caused by endothelial glycocalyx disruption. Recently, recombinant antithrombin (rAT) was reported to protect the endothelial glycocalyx from septic vasculitis; however, the underlying mechanism remains unknown. Here, we investigated the effect of rAT administration on vascular endothelial injury under endotoxemia. Lipopolysaccharide (LPS; 20 mg/kg) was injected intraperitoneally into 10-week-old male C57BL/6 mice, and saline or rAT was administered intraperitoneally at 3 and 24 hours after LPS administration. Subsequently, serum and/or pulmonary tissues were examined for inflammation and cell proliferation and differentiation by histologic, ultrastructural, and microarray analyses. The survival rate was significantly higher in rAT-treated mice than in control mice 48 hours after LPS injection (75% versus 20%; P < 0.05). Serum interleukin-1ß was increased but to a lesser extent in response to LPS injection in rAT-treated mice than in control mice. Lectin staining and ultrastructural studies showed a notable attenuation of injury to the endothelial glycocalyx after rAT treatment. Microarray analysis further showed an up-regulation of gene sets corresponding to DNA repair, such as genes involved in DNA helicase activity, regulation of telomere maintenance, DNA-dependent ATPase activity, and ciliary plasm, after rAT treatment. Thus, rAT treatment may promote DNA repair, attenuate inflammation, and promote ciliogenesis, thereby attenuating the acute respiratory distress syndrome caused by endothelial injury.

Syndecan-1 Depletion Has a Differential Impact on Hyaluronic Acid Metabolism and Tumor Cell Behavior in Luminal and Triple-Negative Breast Cancer Cells.

Glycosaminoglycans (GAGs) and proteoglycans (PGs) are major components of the glycocalyx. The secreted GAG and CD44 ligand hyaluronic acid (HA), and the cell surface PG syndecan-1 (Sdc-1) modulate the expression and activity of cytokines, chemokines, growth factors, and adhesion molecules, acting as critical regulators of tumor cell behavior. Here, we studied the effect of Sdc-1 siRNA depletion and HA treatment on hallmark processes of cancer in breast cancer cell lines of different levels of aggressiveness. We analyzed HA synthesis, and parameters relevant to tumor progression, including the stem cell phenotype, Wnt signaling constituents, cell cycle progression and apoptosis, and angiogenic markers in luminal MCF-7 and triple-negative MDA-MB-231 cells. Sdc-1 knockdown enhanced HAS-2 synthesis and HA binding in MCF-7, but not in MDA-MB-231 cells. Sdc-1-depleted MDA-MB-231 cells showed a reduced CD24-/CD44+ population. Furthermore, Sdc-1 depletion was associated with survival signals in both cell lines, affecting cell cycle progression and apoptosis evasion. These changes were linked to the altered expression of KLF4, MSI2, and miR-10b and differential changes in Erk, Akt, and PTEN signaling. We conclude that Sdc-1 knockdown differentially affects HA metabolism in luminal and triple-negative breast cancer model cell lines and impacts the stem phenotype, cell survival, and angiogenic factors.

Impact of Anesthetic Agents on Endothelial Glycocalyx Injury during Total Knee Arthroplasty: Desflurane- vs. Propofol-Based Anesthesia-A Prospective Randomized Controlled Trial.

BACKGROUND: Ischemia-reperfusion injury and inflammation after tourniquet deflation in total knee arthroplasty are known to be associated with endothelial glycocalyx (EG) injury. This study is aimed at comparing EG injury between desflurane- and propofol-based anesthesia in patients undergoing total knee arthroplasty. MATERIALS AND METHODS: Patients were allocated to the desflurane group or propofol group. The opioid remifentanil was administered intraoperatively in both groups. Blood samples were obtained from the arterial line preoperatively, immediately before and 5 min after tourniquet deflation, and at 1, 6, and 24 h, postoperatively. Serum syndecan-1, cytokines (interleukin-1ß, 6, 10, and tumour necrosis factor-α), and other laboratory values were investigated. RESULTS: Eighty patients were included in the final analysis. The change in syndecan-1 did not significantly differ between the desflurane and propofol groups (peak median level of syndecan-1; 754.5 pg/ml vs. 780.3 pg/ml, respectively, P = 0.512). Laboratory values (serum cytokines, creatinine phosphokinase, lactate dehydrogenase, and lactate levels) were also similar between the two groups. Pulmonary oxygenation was briefly improved after tourniquet deflation in the desflurane group but was similar between the two groups begging at 1 h, postoperatively. CONCLUSIONS: The effect of desflurane was not superior to that of propofol in protecting the EG from ischemia-reperfusion injury during total knee arthroplasty. This trial is registered with Trial Registry Number NCT02756715 (http://clinicaltrials.gov).

The Role of Heparin and Glycocalyx in Blood-Brain Barrier Dysfunction.

The blood-brain barrier (BBB) functions as a dynamic boundary that protects the central nervous system from blood and plays an important role in maintaining the homeostasis of the brain. Dysfunction of the BBB is a pathophysiological characteristic of multiple neurologic diseases. Glycocalyx covers the luminal side of vascular endothelial cells(ECs). Damage of glycocalyx leads to disruption of the BBB, while inhibiting glycocalyx degradation maintains BBB integrity. Heparin has been recognized as an anticoagulant and it protects endothelial glycocalyx from destruction. In this review, we summarize the role of glycocalyx in BBB formation and the therapeutic potency of heparin to provide a theoretical basis for the treatment of neurological diseases related to BBB breakdown.

Changes in endothelial glycocalyx layer protective ability after inflammatory stimulus.

Leukocyte adhesion to the endothelium is an important early step in the initiation and progression of sepsis. The endothelial glycocalyx layer (EGL) has been implicated in neutrophil adhesion and barrier dysfunction, but studies in this area are few. In this report, we examine the hypothesis that damage to the structure of the EGL caused by inflammation leads to increased leukocyte adhesion and endothelial barrier dysfunction. We used human umbilical vein endothelial cells enzymatically treated to remove the EGL components hyaluronic acid (HA) and heparan sulfate (HS) as a model for EGL damage. Using atomic force microscopy, we show reductions in EGL thickness after removal of either HA or HS individually, but the largest decrease, comparable with TNF-α treatment, was observed when both HA and HS were removed. Interestingly, removal of HS or HA individually did not affect neutrophil adhesion significantly, but removal of both constituents resulted in increased neutrophil adhesion. To test EGL contributions to endothelial barrier properties, we measured transendothelial electrical resistance (TEER) and diffusion of fluorescently labeled dextran (10 kDa molecular weight) across the monolayer. Removal of EGL components decreased TEER but had an insignificant effect on dextran diffusion rates. The reduction in TEER suggests that disruption of the EGL may predispose endothelial cells to increased rates of fluid leakage. These data support the view that damage to the EGL during inflammation has significant effects on the accessibility of adhesion molecules, likely facilitates leukocyte adhesion, and may also contribute to increased rates of fluid transport into tissues.