Glycocalyx Components Research Articles

The Glycocalyx and Its Role in Vascular Physiology and Vascular Related Diseases.

PurposeIn 2007 the two senior authors wrote a review on the structure and function of the endothelial glycocalyx layer (Weinbaum in Annu Rev Biomed Eng 9:121–167, 2007). Since then there has been an explosion of interest in this hydrated gel-like structure that coats the luminal surface of endothelial cells that line our vasculature due to its important functions in (A) basic vascular physiology and (B) vascular related diseases. This review will highlight the major advances that have occurred since our 2007 paper.MethodsA literature search mainly focusing on the role of the glycocalyx in the two major areas described above was performed using electronic databases.ResultsIn part (A) of this review, the new formulation of the century old Starling principle, now referred to as the Michel–Weinbaum glycoclayx model or revised Starling hypothesis, is described including new subtleties and physiological ramifications. New insights into mechanotransduction and release of nitric oxide due to fluid shear stress sensed by the glycocalyx are elaborated. Major advances in understanding the organization and function of glycocalyx components, and new techniques for measuring both its thickness and spatio-chemical organization based on super resolution, stochastic optical reconstruction microscopy (STORM) are presented. As discussed in part (B) of this review, it is now recognized that artery wall stiffness associated with hypertension and aging induces glycocalyx degradation, endothelial dysfunction and vascular disease. In addition to atherosclerosis and cardiovascular diseases, the glycocalyx plays an important role in lifestyle related diseases (e.g., diabetes) and cancer. Infectious diseases including sepsis, Dengue, Zika and Corona viruses, and malaria also involve the glycocalyx. Because of increasing recognition of the role of the glycocalyx in a wide range of diseases, there has been a vigorous search for methods to protect the glycocalyx from degradation or to enhance its synthesis in disease environments.ConclusionAs we have seen in this review, many important developments in our basic understanding of GCX structure, function and role in diseases have been described since the 2007 paper. The future is wide open for continued GCX research.

Anisodamine Hydrobromide Protects Glycocalyx and Against the Lipopolysaccharide-Induced Increases in Microvascular Endothelial Layer Permeability and Nitric Oxide Production.

Anisodamine hydrobromide (Ani HBr) has been used to improve the microcirculation during cardiovascular disorders and sepsis. Glycocalyx plays an important role in preserving the endothelial cell (EC) barrier permeability and nitric oxide (NO) production. We aimed to test the hypothesis that Ani HBr could protect the EC against permeability and NO production via preventing glycocalyx shedding. A human cerebral microvascular EC hCMEC/D3 injury model induced by lipopolysaccharide (LPS) was established. Ani HBr was administrated to ECs with the LPS challenge. Cell viability was performed by Cell Counting Kit-8 assay. Cell proliferation and apoptosis were detected by EdU and Hoechst 33342 staining. Apoptosis and cell cycle were also assessed by flow cytometry with annexin V staining and propidium iodide staining, respectively. Then, adherens junction integrity was evaluated basing on the immunofluorescence staining of vascular endothelial cadherin (VE-cadherin). The glycocalyx component heparan sulfate (HS) was stained in ECs. The cell permeability was evaluated by leakage of fluorescein isothiocyanate (FITC)-dextran. Cellular NO production was measured by the method of nitric acid reductase. Ani HBr at 20 μg/mL significantly increased the viability of ECs with LPS challenge, but significantly inhibited the cell viability at 80 μg/mL, showing a bidirectional regulation of cell viability by Ani HBr. Ani HBr had not significantly change the LPS-induced EC proliferation. Ani HBr significantly reversed the induction of LPS on EC apoptosis. Ani HBr reinstated the LPS-induced glycocalyx and VE-cadherin shedding and adherens junction disruption. Ani HBr significantly alleviated LPS-induced EC layer permeability and NO production. Ani HBr protects ECs against LPS-induced increase in cell barrier permeability and nitric oxide production via preserving the integrity of glycocalyx. Ani HBr is a promising drug to rescue or protect the glycocalyx.

Elevated plasma free sialic acid levels in individuals with reduced glomerular filtration rates.

N-acetylneuraminic acid (Neu5Ac, sialic acid) is a negatively charged monosaccharide, and the predominant form of sialic acid in human cells (1). Sialic acid is typically found as the terminal monosaccharide on glycoconjugates, where it plays a role in various physiologic and pathologic interactions (1). Sialylated glycoconjugates are critical contributors to the polyanionic component of the glomerular glycocalyx, contributing to size and charge selectivity for plasma macromolecules (1,2). Podocyte foot process morphology is maintained by the anionic charged sialic acid residues on glycoconjugates in podocyte membranes (2). Free sialic acid is filtered but not reabsorbed by the human kidney, in a fashion similar to that for creatinine (3), but in contrast to the handling of other monosaccharides such as glucose, mannose, galactose, and fructose that are reabsorbed by tubular cells (4). Circulating sialic acid levels, both unbound and bound to glycoconjugates, have only been sporadically studied in different conditions, including some renal disorders (3,5−9); a possible causative link to reduced eGFRs has been seldom discussed or investigated. We designed this study to establish a correlation between eGFR and plasma free sialic acid across a range of subjects with glomerular disorders. This would not only emphasize this often-overlooked aspect of renal filtering of free sialic acid, but also inform our developmental program for glomerular diseases using the sialic acid precursor, N-acetylmannosamine (ManNAc) (10), which was expected to significantly increase plasma free sialic acid levels in subjects with reduced eGFR. Peripheral blood was obtained from eight subjects enrolled in National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) natural history study 94-DK-0127 (ClinicalTrials.gov identifier, NT1392), “Pathogenesis of FSGS,” and from eight subjects enrolled in NIDDK study 16-DK-0036 (ClinicalTrials.gov identifier, NCT02639260), “A Phase 1 Multiple Ascending Dose Study to Evaluate the Safety, Tolerability, and Pharmacokinetics of …

The Role of Proteoglycans in Cancer Metastasis and Circulating Tumor Cell Analysis.

Circulating tumor cells (CTCs) are accessible by liquid biopsies via an easy blood draw. They represent not only the primary tumor site, but also potential metastatic lesions, and could thus be an attractive supplement for cancer diagnostics. However, the analysis of rare CTCs in billions of normal blood cells is still technically challenging and novel specific CTC markers are needed. The formation of metastasis is a complex process supported by numerous molecular alterations, and thus novel CTC markers might be found by focusing on this process. One example of this is specific changes in the cancer cell glycocalyx, which is a network on the cell surface composed of carbohydrate structures. Proteoglycans are important glycocalyx components and consist of a protein core and covalently attached long glycosaminoglycan chains. A few CTC assays have already utilized proteoglycans for both enrichment and analysis of CTCs. Nonetheless, the biological function of proteoglycans on clinical CTCs has not been studied in detail so far. Therefore, the present review describes proteoglycan functions during the metastatic cascade to highlight their importance to CTCs. We also outline current approaches for CTC assays based on targeting proteoglycans by their protein cores or their glycosaminoglycan chains. Lastly, we briefly discuss important technical aspects, which should be considered for studying proteoglycans.

Plasma Endothelial Glycocalyx Components as a Potential Biomarker for Predicting the Development of Disseminated Intravascular Coagulation in Patients With Sepsis.

Sepsis coagulopathy or disseminated intravascular coagulation (DIC) mainly due to progressive endothelial disruption and damage. The glycocalyx is expressed on the endothelial cell surface and contributes to anti-thrombogenicity, anti-inflammatory, and regulates vascular permeability. We aimed to evaluate the clinical utility of plasma glycocalyx components as biomarkers in predicting the onset of DIC in sepsis. This was a prospective observational study of 45 patients with sepsis (June to December 2018). Demographic, clinical (Acute Physiology, Age, Chronic Health Evaluation II [APACHE II], Sequential Organ Failure Assessment [SOFA]), and laboratory data from medical records were analyzed. Endothelial glycocalyx components (syndecan-1, heparan sulfate, hyaluronan) were measured using an ELISA kit. Among the 45 patients (23, sepsis; 22, septic shock), plasma syndecan-1, heparan sulfate, and hyaluronan levels were higher in those with septic shock and were positively correlated with disease severity as determined by the APACHE II and SOFA scores and lactate levels. Receiver operating characteristic curve analysis revealed high sensitivity and specificity of syndecan-1 for predicting septic shock. Further, these levels were compared between patients with or without the development of DIC. Plasma syndecan-1 and hyaluronan levels were significantly elevated in patients with DIC compared to those in patients without DIC and were strongly associated with activated partial thromboplastin time, prothrombin time, and platelet counts. Area under the curve values for predicting DIC based on syndecan-1 and hyaluronan levels measurements were 0.774 and 0.740, respectively. Increased plasma syndecan-1 and hyaluronan levels may be indicators of disease severity and useful predictors for DIC development in sepsis.

Recombinant thrombomodulin protects against LPS-induced acute respiratory distress syndrome via preservation of pulmonary endothelial glycocalyx.

Background and PurposeDisruption of the endothelial glycocalyx is causally related to microvascular endothelial dysfunction, a characteristic of sepsis‐induced acute respiratory distress syndrome (ARDS). Recombinant human thrombomodulin (rhTM) attenuates vascular endothelial injuries, but the underlying mechanism remains elusive. Here, we investigated the structural basis and molecular mechanisms of rhTM effects on vascular endothelial injury in a model of sepsis.Experimental ApproachLPS (20 mg·kg−1) was intraperitoneally injected into 10‐week‐old male C57BL6 mice, and saline or rhTM was intraperitoneally injected 3 and 24 h after LPS injection. Using serum and/or lung tissue, histological, ultrastructural, and microarray analyses were performed.Key ResultsSurvival rate of rhTM‐treated mice was significantly higher than that of control mice 48 h after LPS injection. Serum concentrations of IL‐6 and high‐mobility group box 1 were lower in the rhTM‐treated group than in the control. Injury to the endothelial glycocalyx in pulmonary capillaries was attenuated by rhTM treatment. Gene set enrichment analysis revealed up‐regulation of gene sets corresponding to cell proliferation/differentiation and anti‐inflammation, such as the TGF‐β pathway, and negative regulation of IL‐6, upon rhTM treatment. Gene expression of heparan sulfate 6‐O‐sulfotransferase 1 and endothelial cell‐specific molecule 1 (components of the endothelial glycocalyx) was significantly preserved by rhTM treatment, and their protein expression levels were maintained in endothelial cells.Conclusion and ImplicationsOur findings show that rhTM treatment affected inflammation, cell proliferation/differentiation, and glycocalyx synthesis in serum and lung tissue, subsequently attenuating ARDS caused by endothelial injury.

Elements of the Endomucin Extracellular Domain Essential for VEGF-Induced VEGFR2 Activity.

Endomucin (EMCN) is the type I transmembrane glycoprotein, mucin-like component of the endothelial cell glycocalyx. We have previously shown that EMCN is necessary for vascular endothelial growth factor (VEGF)-induced VEGF receptor 2 (VEGFR2) internalization and downstream signaling. To explore the structural components of EMCN that are necessary for its function and the molecular mechanism of EMCN in VEGF-induced endothelial functions, we generated a series of mouse EMCN truncation mutants and examined their ability to rescue VEGF-induced endothelial functions in human primary endothelial cells (EC) in which endogenous EMCN had been knocked down using siRNA. Expression of the mouse full-length EMCN (FL EMCN) and the extracellular domain truncation mutants ∆21-81 EMCN and ∆21-121 EMCN, but not the shortest mutant ∆21-161 EMCN, successfully rescued the VEGF-induced EC migration, tube formation, and proliferation. ∆21-161 EMCN failed to interact with VEGFR2 and did not facilitate VEGFR2 internalization. Deletion of COSMC (C1GalT1C1) revealed that the abundant mucin-type O-glycans were not required for its VEGFR2-related functions. Mutation of the two N-glycosylation sites on ∆21-121 EMCN abolished its interaction with VEGFR2 and its function in VEGFR2 internalization. These results reveal ∆21-121 EMCN as the minimal extracellular domain sufficient for VEGFR2-mediated endothelial function and demonstrate an important role for N-glycosylation in VEGFR2 interaction, internalization, and angiogenic activity.

An Acquired and Endogenous Glycocalyx Forms a Bidirectional ‘Don't Eat’ and ‘Don't Eat Me’ Barrier to Phagocytosis

Macrophages continuously survey their environment in search of pathogens or apoptotic corpses or debris. Targets intended for clearance expose ligands that initiate their phagocytosis (“eat me” signals), while others avoid phagocytosis by displaying inhibitory ligands (“don’t eat me” signals). We report that such ligands can be obscured by the glycosaminoglycans and glycoproteins that coat pathogenic as well as malignant phagocytic targets. In addition, a reciprocal barrier of self-synthesized or acquired glycocalyx components on the macrophage surface shrouds phagocytic receptors, curtailing their ability to engage particles. The coating layers of macrophages and their targets hinder phagocytosis by both steric and electrostatic means. Their removal by enzymatic means is shown to markedly enhance phagocytic efficiency. In particular, we show that removal of mucins, which are overexpressed in cancer cells, facilitates their clearance. These results shed light on the physical barriers that modulate phagocytosis, which have been heretofore underappreciated.

The Beneficial Effect of HES on Vascular Permeability and Its Relationship With Endothelial Glycocalyx and Intercellular Junction After Hemorrhagic Shock.

BackgroundVascular leakage is a common complication of hemorrhagic shock. Endothelial glycocalyx plays a crucial role in the protection of vascular endothelial barrier function. Hydroxyethyl starch (HES) is a commonly used resuscitation fluid for hemorrhagic shock. However, whether the protective effect of HES on vascular permeability after hemorrhagic shock is associated with the endothelial glycocalyx is unclear.MethodsUsing hemorrhagic shock rat model and hypoxia treated vascular endothelial cells (VECs), effects of HES (130/0.4) on pulmonary vascular permeability and the relationship to endothelial glycocalyx were observed.ResultsPulmonary vascular permeability was significantly increased after hemorrhagic shock, as evidenced by the increased permeability of pulmonary vessels to albumin-fluorescein isothiocyanate conjugate (FITC-BSA) and Evans blue, the decreased transendothelial electrical resistance of VECs and the increased transmittance of FITC-BSA. The structure of the endothelial glycocalyx was destroyed, showing a decrease in thickness. The expression of heparan sulfate, hyaluronic acid, and chondroitin sulfate, the components of the endothelial glycocalyx, was significantly decreased. HES (130/0.4) significantly improved the vascular barrier function, recovered the thickness and the expression of components of the endothelial glycocalyx by down-regulating the expression of heparinase, hyaluronidase, and neuraminidase, and meanwhile increased the expression of intercellular junction proteins ZO-1, occludin, and VE-cadherin. Degradation of endothelial glycocalyx with degrading enzyme (heparinase, hyaluronidase, and neuraminidase) abolished the beneficial effect of HES on vascular permeability, but had no significant effect on the recovery of the expression of endothelial intercellular junction proteins induced by HES (130/0.4). HES (130/0.4) decreased the expression of cleaved-caspase-3 induced by hemorrhagic shock.ConclusionsHES (130/0.4) has protective effect on vascular barrier function after hemorrgic shock.The mechanism is mainly related to the protective effect of HES on endothelial glycocalyx and intercellular junction proteins. The protective effect of HES on endothelial glycocalyx was associated with the down-regulated expression of heparinase, hyaluronidase, and neuraminidase. HES (130/0.4) had an anti-apoptotic effect in hemorrhagic shock.

Endothelial glycocalyx and severity and vulnerability of coronary plaque in patients with coronary artery disease

Background and aimsEndothelial glycocalyx covers the endothelium and maintains vascular integrity. However, its association with the severity and vulnerability of coronary artery disease (CAD) remains to be elucidated. MethodsA total of 259 consecutive patients with stable CAD requiring percutaneous coronary intervention (PCI) were prospectively enrolled. Patients were classified into 2 groups according to the median value of serum syndecan-1, which is a core component of the endothelial glycocalyx (lower syndecan-1 group [syndecan-1 <99.0 ng/mL], n = 130; higher syndecan-1 group [syndecan-1 ≥99.0 ng/mL], n = 129). Severity of CAD and focal plaque vulnerability in culprit lesion were evaluated using angiography and optical coherence tomography. ResultsThere was no significant difference in clinical characteristics between the lower syndecan-1 group and the higher syndecan-1 group other than the prevalence of family history of CAD (19 vs. 32%, p = 0.022), prior PCI history (45 vs. 60%, p = 0.015) and estimated glomerular filtration rate (57.8 ± 17.2 vs. 50.9 ± 25.6 ml/min/1.73 m2, p = 0.011). Although disease severity on angiogram was comparable between the 2 groups, the prevalence of lipid-rich plaque (40 vs. 19%, p = 0.004) and thin-cap fibroatheroma (20 vs. 6%, p = 0.006) was significantly higher in the lower syndecan-1 group than the higher syndecan-1 group. Lower syndecan-1 level was independently associated with higher prevalence of lipid-rich plaque (odds ratio 3.626, 95% confidence interval 1.535–8.566, p = 0.003). ConclusionsLower syndecan-1 level was associated with higher prevalence of vulnerable plaque in patients with CAD. This finding suggests the association between impaired endothelial glycocalyx and the development of vulnerable plaque.

Heparan Sulfate Modulates Heart Vascular Remodeling Induced by S. Aureus Sepsis

Sepsis is a life‐threatening condition triggered by a dysregulated host response to microbial infection resulting in vascular dysfunction and organ failure. Infection by Staphylococcus aureus infections can result in sepsis, which is correlated with remodeling of the vascular glycocalyx. Heparan sulfate (HS), a major component of the vascular glycocalyx, undergoes shedding during sepsis, but the impact of vascular HS and shedding on sepsis outcomes is unknown. In this study, we modified the composition of HS in endothelia cells in mice by crossbreeding animals bearing a conditional allele in N‐acetylglucosamine N‐deacetylase‐N‐sulfotransferase‐1 (Ndst1f/f) with mice bearing the Cre recombinase driven by the Tie2 promoter (Tie2‐Cre). Ndst1f/fTie2Cre mice exhibit HS with overall reduced sulfation in endothelial and myeloid cells. Ndst1f/fTie2Cre mice succumbed earlier to sepsis induced by S. aureus. Application of an in vivo vascular tagging method revealed drastic proteomic changes in the hearts of Ndst1f/fTie2Cre mice, including increased markers of cardiac damage. These studies provide a link between vascular heparan sulfate and early death due to S. aureus infection. Additional measures of characterizing cardiac damage will further explore the heart proteomics signature in Ndst1f/fTie2Cre mice to increased S. aureus susceptibility.

Sulfation of endothelial heparan sulfate mediates Staphylococcus aureus induced hepatotoxicity

Sepsis is defined as life‐threatening organ dysfunction caused by a dysregulated host response to infection and vascular pathology. Understanding the linkage of vascular pathology to organ failure could provide a strategy for treating the disease. Heparan sulfate proteoglycans are critical components of the vascular endothelial glycocalyx and play a key role in sepsis outcome in mice. To study how heparan sulfate modulates organ‐specific vascular pathology during sepsis, we induced Staphylococcus aureus sepsis in Ndst1f/fTie2Cre mice, which express under‐sulfated heparan sulfate in endothelial and myeloid tissues. Hepatic coagulopathy and infarction induced by S. aureus was reduced in Ndst1f/fTie2Cre mice, but not in Ndst1f/fLysMCre or Ndst1f/fPF4Cre mice. To examine how modifying heparan sulfate structure mitigates hepatic damage, pro‐inflammatory neutrophil trafficking and behavior was monitored in the liver during sepsis. Ndst1f/fTie2Cre mice contained fewer extravasated neutrophils in the liver early in sepsis. Intravital microscopy in the liver further verified that Ndst1f/fTie2Cre mice have neutrophil trafficking deficiencies after both sterile and septic injury. Further studies will focus on the role of heparan sulfate in neutrophil trafficking and downstream neutrophil‐driven vascular dysfunction in sepsis.Support or Funding InformationThis work is supported by NIH P01 HL131474 and the UCSD Microbial Sciences Graduate Research Initiative Fellowship.

The influence of hyperglycemia on neutrophil extracellular trap formation and endothelial glycocalyx damage in a mouse model of type 2 diabetes.

Hyperglycemia induces vascular dysfunction that is thought to be initiated by neutrophils. Neutrophil activation produces endothelial injury by pathways that include NETosis, a type of specific cell death. In this study, we investigated the effects of hyperglycemia on neutrophil activation, cell death, NETosis, and endothelial glycocalyx damage using a mouse diabetes model. We used db/db mice as a type 2 diabetes model, and C57BL/6 mice were the controls. At 5, 8, and 12weeks of age, the proportion of CD11b+ granulocytes/monocytes, neutrophil extracellular trap (NET)-forming granulocytes/monocytes, and damaged and nonviable granulocytes/monocytes was analyzed. In addition, serum levels of high mobility group box 1, histone H3, and glycocalyx components that included syndecan-1 and hyaluronan were measured. In diabetic mice, we observed an increased proportion of CD11b+ granulocytes/monocytes. The proportion of NET-forming granulocytes/monocytes increased from the early stages of the experiments. The proportions of damaged and nonviable granulocytes/monocytes increased over time. In the 12-week-old diabetic mice, serum histone H3 levels increased. Circulating levels of syndecan-1 and hyaluronan decreased over time and were lower in diabetic mice. Neutrophil activation and cell death induce endothelial glycocalyx damage, and NET formation also participates in the mechanisms of vascular injury in type 2 diabetes.

Rapid Ascent to 4559 m Is Associated with Increased Plasma Components of the Vascular Endothelial Glycocalyx and May Be Associated with Acute Mountain Sickness.

Background: The stress of high altitude alters vascular permeability, which may be related to structural changes in the endothelial glycocalyx. We aimed to study these changes by measuring plasma concentrations of several glycocalyx components upon exposure to high altitude. Methods: Plasma collected from 17 subjects at low altitude (423 m) and at three time points (7, 20, and 44 hours) after rapid ascent to high altitude (4559 m) were evaluated for concentrations of three glycocalyx components: syndecan-1, intercellular adhesion molecule-1 (ICAM-1), and heparan sulfate. Vital signs and echocardiographic measurement of systolic pulmonary artery pressure (sPAP) and cardiac output were also obtained at low and high altitudes. Results: Mean age of the study population was 35.5 ± 11.2 years with a body mass index of 22.7 ± 2.5 kg/m2. Concentrations of ICAM-1 and heparan sulfate increased from baseline to 7 hours after arrival at high altitude; the ICAM-1 rise persisted at 20 hours. Syndecan-1 concentrations were increased only at 44 hours. Increased ICAM-1 concentrations correlated with sPAP and peripheral edema. Elevations in heparan sulfate appeared to correlate with acute mountain sickness (AMS). Conclusions: Levels of circulating glycocalyx components increase after exposure to high altitude and may correlate with AMS. Measuring plasma concentrations of various glycocalyx components could serve as a useful tool for further evaluation of vascular endothelial injury and repair in illness at high altitude.

87 Evaluating Endothelial Dysfunction in Thermally-injured Patients with syndecan-1, Tissue Factor Pathway Inhibitor, and Thrombomodulin as Predictors of Mortality

Abstract Introduction Endotheliopathy in burn patients is largely uncharacterized. Syndecan-1 (SDC-1), thrombomodulin (TM), and tissue factor pathway inhibitor (TFPI) are components of the vascular endothelial glycocalyx. Proteolytic cleavage of these moieties may yield biomarkers for endothelial damage. The aim of this study is to evaluate endotheliopathy after burn injury by monitoring plasma levels of these biomarkers over time to investigate potential relationship to mortality. Methods Burn injured patients presenting to a regional burn center from 2012 to 2017 were prospectively enrolled. Blood samples were collected at 0, 2, 4, 8, 12, 24, 36, 48, 60, and 72 hours from admission. Plasma SDC-1, TM, and TFPI levels were quantified by ELISA. Demographic data and injury characteristics were obtained from the medical chart. Patients with concomitant inhalation injury, trauma, or &amp;lt; 10% total body surface area (TBSA) burns were excluded. Statistical analysis was performed using mixed-effect models with Sidak’s correction for multiple comparisons. Significance was set at p =0.05. Data are presented as mean ± standard deviation. Results A cohort of 22 patients was identified with an average age of 45±14 years, TBSA of 30±15%, with 6 patients who died from their injuries. The deceased group was older (59±13 vs. 40±10 years, p = 0.01), and there was no significant difference in burn size. Mean SDC-1 levels were higher in the deceased group at all time points (p=0.0004) and this difference was significant at hour 12 (106±11 vs. 41±31 ng/mL, p = 0.0002), hour 24 (160±39 vs. 35±20 ng/mL, p = 0.04) and hour 72 (100±3 vs. 35±38 ng/mL, p = 0.01). Mean soluble TM levels were higher in the deceased group after hour 12 (p = 0.04) and there was a trend towards higher TFPI levels after hour 12 in the deceased group. Conclusions Biomarkers are elevated in patients following burn injury who die, when inhalation injury and trauma are excluded. Given equivalent TBSA, older patients appear more sensitive to thermally induced glycocalyx degradation. SDC-1 shows the greatest promise as a prognostic indicator as levels tend to be higher among deceased patients on admission and are significantly higher as early as hour 12. Applicability of Research to Practice Reliable assessment of the patient’s endothelial damage may hold predictive value for clinicians and could assist in clinical decision making. Further research must investigate endotheliopathy in burn patients.

Disrupted Glycocalyx as a Source of Ocular Surface Biomarkers.

The glycocalyx is a dense and diverse coat of glycans and glycoconjugates responsible for maintaining cell surface integrity and regulating the interaction of cells with the external environment. Transmembrane mucins such as MUC1 and MUC16 comprise a major component of the epithelial glycocalyx and are currently used to monitor disease progression in cancer. At the ocular surface, multiple lines of evidence indicate that abnormal expression of the enzymes responsible for glycan biosynthesis during pathological conditions impairs the glycosylation of transmembrane mucins. It is now becoming clear that these changes contribute to modify the interaction of mucins with galectin-3, a multimeric lectin crucial for preserving the ocular surface epithelial barrier. This review highlights the potential of using the epithelial glycocalyx as a reliable source for the generation of biomarkers to diagnose and monitor ocular surface disease.

In Vivo Imaging of the Buccal Mucosa Shows Loss of the Endothelial Glycocalyx and Perivascular Hemorrhages in Pediatric Plasmodium falciparum Malaria.

Severe malaria is mostly caused by Plasmodium falciparum, resulting in considerable, systemic inflammation and pronounced endothelial activation. The endothelium forms an interface between blood and tissue, and vasculopathy has previously been linked with malaria severity. We studied the extent to which the endothelial glycocalyx that normally maintains endothelial function is involved in falciparum malaria pathogenesis by using incident dark-field imaging in the buccal mucosa. This enabled calculation of the perfused boundary region, which indicates to what extent erythrocytes can permeate the endothelial glycocalyx. The perfused boundary region was significantly increased in severe malaria patients and mirrored by an increase of soluble glycocalyx components in plasma. This is suggestive of a substantial endothelial glycocalyx loss. Patients with severe malaria had significantly higher plasma levels of sulfated glycosaminoglycans than patients with uncomplicated malaria, whereas other measured glycocalyx markers were raised to a comparable extent in both groups. In severe malaria, the plasma level of the glycosaminoglycan hyaluronic acid was positively correlated with the perfused boundary region in the buccal cavity. Plasma hyaluronic acid and heparan sulfate were particularly high in severe malaria patients with a low Blantyre coma score, suggesting involvement in its pathogenesis. In vivo imaging also detected perivascular hemorrhages and sequestering late-stage parasites. In line with this, plasma angiopoietin-1 was decreased while angiopoietin-2 was increased, suggesting vascular instability. The density of hemorrhages correlated negatively with plasma levels of angiopoietin-1. Our findings indicate that as with experimental malaria, the loss of endothelial glycocalyx is associated with vascular dysfunction in human malaria and is related to severity.

3D Cell-Culture Models for the Assessment of Anticoagulant and Anti-Inflammatory Properties of Endothelial Cells.

Endothelial cells (EC) play a crucial role in the pathophysiology of cardiovascular diseases, ischemia/reperfusion injury, and graft rejection in (xeno-)transplantation. In such nonphysiological conditions, EC are known to lose their quiescent phenotype and switch into an actively pro-inflammatory, procoagulant, and anti-fibrinolytic state. This case happens essentially because the endothelial glycocalyx-a layer of proteoglycans and glycoproteins covering the luminal surface of the endothelium-is shed. Heparan sulfate, one of the main components of the endothelial glycocalyx, contributes to its negative charge. In addition, many plasma proteins such as antithrombin III, superoxide dismutase, C1 inhibitor, and growth factors and cytokines bind to heparan sulfate and by this scenario contribute to the establishment of an anticoagulant and anti-inflammatory endothelial surface. Shedding of the glycocalyx results in a loss of plasma proteins from the endothelial surface, and this phenomenon causes the switch in phenotype. Particularly in xenotransplantation, both hyperacute and acute vascular rejection are characterized by coagulation dysregulation, a situation in which EC are the main players.Since many years, EC have been used in vitro in 2D flatbed cell culture models, with or without the application of shear stress. Such models have also been used to assess the effect of human transgenes on complement- and coagulation-mediated damage of porcine EC in the context of xenotransplantation. The methods described in this chapter include the analysis of endothelial cell-blood interactions without the necessity of using anticoagulants as the increased EC surface-to-volume ratio allows for natural anticoagulation of blood. Furthermore, this chapter contains the description of a novel microfluidic in vitro model carrying important features of small blood vessels, such as a 3D round-section geometry, shear stress, and pulsatile flow-all this in a closed circuit, recirculating system aiming at reproducing closely the in vivo situation in small vessels.

Hemodilution causes glycocalyx shedding without affecting vascular endothelial barrier permeability in rats

The consequences of acute normovolemic hemodilution (ANH) following different types of fluids on the different components of the glycocalyx and on vascular barrier permeability (VBP) remain unknown. The aim of the study was to investigate whether the microcirculatory disruption and glycocalyx shedding induced by ANH alters VBP and whether this is affected by the composition and volume of the resuscitation fluid. Anesthetized Wistar albino rats (n=24) underwent stepwise ANH at hematocrit levels of 35%, 25%, 20%, and 15% induced by the exchange of blood with 6% balanced hydroxyethyl starch (1:1), balanced crystalloid (1:3), and normal saline (NS) (1:3). Glycocalyx-shed products were measured at each level of hemodilution. VBP was reflected in the decay of fluorescence dyes of different molecular size and their plasma retention ratios. Edema was assessed by measuring organ water content and muscle microcirculation by hand-held videomicroscopy. NS caused increased degradation of heparan sulfate and hyaluronan compared with the control group (P=0.003, P=0.004, respectively). Neither VBP nor tissue edema was affected by the fluid used. The total and perfused vessel densities within the microcirculation of muscle tissue decreased at hematocrit 15% in the balanced crystalloid (P=0.02) and NS groups only (P<0.0001, P=0.0003, respectively) compared with baseline. Balanced colloid solution preserved the glycocalyx layer better than balanced and unbalanced crystalloid solutions while maintaining the microcirculatory function associated with an improved total intravascular volume. Among the fluids tested, NS caused the most microcirculatory alterations. While ANH caused the degradation of glycocalyx components regardless of fluid, it did not disrupt the vascular barrier as indicated by macromolecular leakage. The results of this study provide insight into the choice of fluid for optimal perioperative fluid management and the consequences of fluid type on the vascular barrier, glycocalyx, and microcirculation.

The stressed vascular barrier and coagulation - The impact of key glycocalyx components on in vitro clot formation

IntroductionA functional vascular barrier controlling leukocyte recruitment into the perivascular space relies on an intact endothelial glycocalyx (EGX). Critical disease states such as sepsis or trauma can induce massive shedding of EGX components into the blood stream. Previous studies have shown that high blood levels of EGX components are correlated with bleeding in patients. The mechanism behind that observation remains to be fully elucidated. Material and methodsThe EGX components syndecan-1 (S1), hyaluronic acid (HA) and heparan sulfate (HS) were added to blood samples of 10 healthy male volunteers separately in three distinct concentrations to mimic three severity levels of in vitro EGX shedding. We analyzed spiked blood samples for leukocyte derived reactive oxygen species (ROS) release as a measure for innate immune activation and evaluated the impact on coagulation using clinical standard coagulation tests (SCTs) as well as rotational thrombelastometry (ROTEM®). ResultsWhereas ROS formation by polymorphonuclear leukocytes (PMN) was unaltered by all three substances, high concentrations of HS showed prolonged aPTT and TT compared to controls and S1 or HA. Changes in ROTEM® were discrete and mostly within normal range of values but analyses showed a significant reduction of clot firmness and formation by all EGX components compared to controls. Furthermore, alterations by HA and HS were dose dependent. Only HS showed a heparin like effect supporting the findings of SCTs. ConclusionsAll EGX components interfere with clot formation and strength. HS mimics heparin effects in ROTEM® that confirm detectable alterations of standard coagulation tests.