Penetration of Cell Surface Glycocalyx by Enveloped Viruses Is Aided by Weak Multivalent Adhesive Interaction.

Viral infection usually begins with adhesion between the viral particle and viral receptors displayed on the cell membrane. The exterior surface of the cell membrane is typically coated with a brush-like layer of molecules, the glycocalyx, that the viruses need to penetrate. Although there is extensive literature on the biomechanics of virus-cell adhesion, much of it is based on continuum-level models that do not address the question of how virus/cell-membrane adhesion occurs through the glycocalyx. In this work, we present a simulation study of the penetration mechanism. Using a coarse-grained molecular model, we study the force-driven and diffusive penetration of a brush-like glycocalyx by viral particles. For force-driven penetration, we find that viral particles smaller than the spacing of molecules in the brush reach the membrane surface readily. For a given maximum force, viral particles larger than the minimum spacing of brush molecules arrest at some distance from the membrane, governed by the balance of elastic and applied forces. For the diffusive case, we find that weak but multivalent attraction between the glycocalyx molecules and the virus effectively leads to its engulfment by the glycocalyx. Our finding provides potential guidance for developing glycocalyx-targeting drugs and therapies by understanding how virus-cell adhesion works.

Extensive blood transcriptome analysis reveals cellular signaling networks activated by circulating glycocalyx components reflecting vascular injury in COVID-19.

Background: Degradation of the endothelial protective glycocalyx layer during COVID-19 infection leads to shedding of major glycocalyx components. These circulating proteins and their degradation products may feedback on immune and endothelial cells and activate molecular signaling cascades in COVID-19 associated microvascular injury. To test this hypothesis, we measured plasma glycocalyx components in patients with SARS-CoV-2 infection of variable disease severity and identified molecular signaling networks activated by glycocalyx components in immune and endothelial cells. Methods: We studied patients with RT-PCR confirmed COVID-19 pneumonia, patients with COVID-19 Acute Respiratory Distress Syndrome (ARDS) and healthy controls (wildtype, n=20 in each group) and measured syndecan-1, heparan sulfate and hyaluronic acid. The in-silico construction of signaling networks was based on RNA sequencing (RNAseq) of mRNA transcripts derived from blood cells and of miRNAs isolated from extracellular vesicles from the identical cohort. Differentially regulated RNAs between groups were identified by gene expression analysis. Both RNAseq data sets were used for network construction of circulating glycosaminoglycans focusing on immune and endothelial cells. Results: Plasma concentrations of glycocalyx components were highest in COVID-19 ARDS. Hyaluronic acid plasma levels in patients admitted with COVID-19 pneumonia who later developed ARDS during hospital treatment (n=8) were significantly higher at hospital admission than in patients with an early recovery. RNAseq identified hyaluronic acid as an upregulator of TLR4 in pneumonia and ARDS. In COVID-19 ARDS, syndecan-1 increased IL-6, which was significantly higher than in pneumonia. In ARDS, hyaluronic acid activated NRP1, a co-receptor of activated VEGFA, which is associated with pulmonary vascular hyperpermeability and interacted with VCAN (upregulated), a proteoglycan important for chemokine communication. Conclusions: Circulating glycocalyx components in COVID-19 have distinct biologic feedback effects on immune and endothelial cells and result in upregulation of key regulatory transcripts leading to further immune activation and more severe systemic inflammation. These consequences are most pronounced during the early hospital phase of COVID-19 before pulmonary failure develops. Elevated levels of circulating glycocalyx components may early identify patients at risk for microvascular injury and ARDS. The timely inhibition of glycocalyx degradation could provide a novel therapeutic approach to prevent the development of ARDS in COVID-19.

Vascular Endothelial Glycocalyx Damage and Potential Targeted Therapy in COVID-19.

COVID-19 is a highly infectious respiratory disease caused by a new coronavirus known as SARS-CoV-2. COVID-19 is characterized by progressive respiratory failure resulting from diffuse alveolar damage, inflammatory infiltrates, endotheliitis, and pulmonary and systemic coagulopathy forming obstructive microthrombi with multi-organ dysfunction, indicating that endothelial cells (ECs) play a central role in the pathogenesis of COVID-19. The glycocalyx is defined as a complex gel-like layer of glycosylated lipid-protein mixtures, which surrounds all living cells and acts as a buffer between the cell and the extracellular matrix. The endothelial glycocalyx layer (EGL) plays an important role in vascular homeostasis via regulating vascular permeability, cell adhesion, mechanosensing for hemodynamic shear stresses, and antithrombotic and anti-inflammatory functions. Here, we review the new findings that described EGL damage in ARDS, coagulopathy, and the multisystem inflammatory disease associated with COVID-19. Mechanistically, the inflammatory mediators, reactive oxygen species (ROS), matrix metalloproteases (MMPs), the glycocalyx fragments, and the viral proteins may contribute to endothelial glycocalyx damage in COVID-19. In addition, the potential therapeutic strategies targeting the EGL for the treatment of severe COVID-19 are summarized and discussed.

Heparanase Is a Putative Mediator of Endothelial Glycocalyx Damage in COVID-19 - A Proof-of-Concept Study.

Coronavirus disease 2019 (COVID-19) is a systemic disease associated with injury (thinning) of the endothelial glycocalyx (eGC), a protective layer on the vascular endothelium. The aim of this translational study was to investigate the role of the eGC-degrading enzyme heparanase (HPSE), which is known to play a central role in the destruction of the eGC in bacterial sepsis. Excess activity of HPSE in plasma from COVID-19 patients correlated with several markers of eGC damage and perfused boundary region (PBR, an inverse estimate of glycocalyx dimensions of vessels with a diameter 4-25 µm). In a series of translational experiments, we demonstrate that the changes in eGC thickness of cultured cells exposed to COVID-19 serum correlated closely with HPSE activity in concordant plasma samples (R = 0.82, P = 0.003). Inhibition of HPSE by a nonanticoagulant heparin fragment prevented eGC injury in response to COVID-19 serum, as shown by atomic force microscopy and immunofluorescence imaging. Our results suggest that the protective effect of heparin in COVID-19 may be due to an eGC-protective off-target effect.

Endothelial glycocalyx damage in patients with severe COVID-19 on mechanical ventilation - A prospective observational pilot study.

BACKGROUND: Coronavirus disease (COVID-19) associated endotheliopathy and microvascular dysfunction are of concern. OBJECTIVE: The objective of the present single-center observational pilot study was to compare endothelial glycocalyx (EG) damage and endotheliopathy in patients with severe COVID-19 (COVID-19 group) with patients with bacterial pneumonia with septic shock (non-COVID group). METHODS: Biomarkers of EG damage (syndecan-1), endothelial cells (EC) damage (thrombomodulin), and activation (P-selectin) were measured in blood on three consecutive days from admission to the intensive care unit (ICU). The sublingual microcirculation was studied by Side-stream Dark Field (SDF) imaging with automatic assessment. RESULTS: We enrolled 13 patients in the non-COVID group (mean age 70 years, 6 women), and 15 in the COVID-19 group (64 years old, 3 women). The plasma concentrations of syndecan-1 were significantly higher in the COVID-19 group during all three days. Differences regarding other biomarkers were not statistically significant. The assessment of the sublingual microcirculation showed improvement on Day 2 in the COVID-19 group. Plasma levels of C-reactive protein (CRP) were significantly higher on the first two days in the COVID-19 group. Plasma syndecan-1 and CRP were higher in patients suffering from severe COVID-19 pneumonia compared to bacterial pneumonia patients. CONCLUSIONS: These findings support the role of EG injury in the microvascular dysfunction in COVID-19 patients who require ICU.

Endothelial glycocalyx degradation in multisystem inflammatory syndrome in children related to COVID-19.

Multisystem inflammatory syndrome in children (MIS-C) represents a rare but severe complication of severe acute respiratory syndrome coronavirus 2 infection affecting children that can lead to myocardial injury and shock. Vascular endothelial dysfunction has been suggested to be a common complicating factor in patients with coronavirus disease 2019 (COVID-19). This study aims to characterize endothelial glycocalyx degradation in children admitted with MIS-C. We collected blood and urine samples and measured proinflammatory cytokines, myocardial injury markers, and endothelial glycocalyx markers in 17 children admitted with MIS-C, ten of which presented with inflammatory shock requiring intensive care admission and hemodynamic support with vasopressors. All MIS-C patients presented signs of glycocalyx deterioration with elevated levels of syndecan-1 in blood and both heparan sulfate and chondroitin sulfate in the urine. The degree of glycocalyx shedding correlated with tumor necrosis factor-α concentration. Five healthy age-matched children served as controls. Patients with MIS-C presented severe alteration of the endothelial glycocalyx that was associated with disease severity. Future studies should clarify if glycocalyx biomarkers could effectively be predictive indicators for the development of complications in adult patients with severe COVID-19 and children with MIS-C. KEY MESSAGES : Children admitted with MIS-C presented signs of endothelial glycocalyx injury with elevated syndecan-1 and heparan sulfate level. Syndecan-1 levels were associated with MIS-C severity and correlated TNF-α concentration. Syndecan-1 and heparan sulfate may represent potential biomarkers for patients with severe COVID-19 or MIS-C.

Cerebral microvascular endothelial glycocalyx damage, its implications on the blood-brain barrier and a possible contributor to cognitive impairment.

The socio-economic impact of diseases associated with cognitive impairment is increasing. According to the Alzheimer's Society there are over 850,000 people with dementia in the UK, costing the UK £26 billion in 2013. Therefore, research into treatment of those conditions is vital. Research into the cerebral endothelial glycocalyx (CeGC) could offer effective treatments. The CeGC, consisting of proteoglycans, glycoproteins and glycolipids, is a dynamic structure covering the luminal side oftheendothelial cells of capillaries throughout the body. The CeGC is thicker in cerebral micro vessels, suggesting specialisation for its function as part of the blood-brain barrier (BBB). Recent research evidences that the CeGC is vital in protecting fragile parenchymal tissue and effective functioning of the BBB, as one particularly important CeGC function is to act as a protective barrier and permeability regulator. CeGC degradation is one of the factors which can lead to an increase in BBB permeability. It occurs naturally in aging, nevertheless, premature degradationhas beenevidencedin multipleconditions linked to cognitive impairment, such as inflammation,brain edema, cerebral malaria, Alzheimer's and recently Covid-19. Increasing knowledge of the mechanisms of CeGC damage has led to research into preventative techniques showing that CeGC is a possible diagnostic marker and a therapeutic target. However, the evidence is relatively new, inconsistent and demonstrated mainly in experimental models. This review evaluates the current knowledge of the CeGC, its structure, functions, damage and repair mechanisms and the impact of its degeneration on cognitive impairment in multiple conditions, highlighting the CeGC as a possible diagnostic marker and a potential target for therapeutic treatment.

Syndecan-1, an indicator of endothelial glycocalyx degradation, predicts outcome of patients admitted to an ICU with COVID-19.

BACKGROUND: We investigated the feasibility of two biomarkers of endothelial damage (Syndecan-1 and thrombomodulin) in coronavirus disease 2019 (COVID-19), and their association with inflammation, coagulopathy, and mortality. METHODS: The records of 49 COVID-19 patients who were admitted to an intensive care unit (ICU) in Wuhan, China between February and April 2020 were examined. Demographic, clinical, and laboratory data, and outcomes were compared between survivors and non-survivors COVID-19 patients, and between patients with high and low serum Syndecan-1 levels. The dynamics of serum Syndecan-1 levels were also analyzed. RESULTS: The levels of Syndecan-1 were significantly higher in non-survivor group compared with survivor group (median 1031.4 versus 504.0 ng/mL, P = 0.002), and the levels of thrombomodulin were not significantly different between these two groups (median 4534.0 versus 3780.0 ng/mL, P = 0.070). Kaplan-Meier survival analysis showed that the group with high Syndecan-1 levels had worse overall survival (log-rank test: P = 0.023). Patients with high Syndecan-1 levels also had significantly higher levels of thrombomodulin, interleukin-6, and tumor necrosis factor-α. Data on the dynamics of Syndecan-1 levels indicated much greater variations in non-survivors than survivors. CONCLUSIONS: COVID-19 patients with high levels of Syndecan-1 develop more serious endothelial damage and inflammatory reactions, and have increased mortality. Syndecan-1 has potential for use as a marker for progression or severity of COVID-19. Protecting the glycocalyx from destruction is a potential treatment for COVID-19.

Pathogenesis of COVID-19 described through the lens of an undersulfated and degraded epithelial and endothelial glycocalyx.

The glycocalyx surrounds every eukaryotic cell and is a complex mesh of proteins and carbohydrates. It consists of proteoglycans with glycosaminoglycan side chains, which are highly sulfated under normal physiological conditions. The degree of sulfation and the position of the sulfate groups mainly determine biological function. The intact highly sulfated glycocalyx of the epithelium may repel severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) through electrostatic forces. However, if the glycocalyx is undersulfated and 3-O-sulfotransferase 3B (3OST-3B) is overexpressed, as is the case during chronic inflammatory conditions, SARS-CoV-2 entry may be facilitated by the glycocalyx. The degree of sulfation and position of the sulfate groups will also affect functions such as immune modulation, the inflammatory response, vascular permeability and tone, coagulation, mediation of sheer stress, and protection against oxidative stress. The rate-limiting factor to sulfation is the availability of inorganic sulfate. Various genetic and epigenetic factors will affect sulfur metabolism and inorganic sulfate availability, such as various dietary factors, and exposure to drugs, environmental toxins, and biotoxins, which will deplete inorganic sulfate. The role that undersulfation plays in the various comorbid conditions that predispose to coronavirus disease 2019 (COVID-19), is also considered. The undersulfated glycocalyx may not only increase susceptibility to SARS-CoV-2 infection, but would also result in a hyperinflammatory response, vascular permeability, and shedding of the glycocalyx components, giving rise to a procoagulant and antifibrinolytic state and eventual multiple organ failure. These symptoms relate to a diagnosis of systemic septic shock seen in almost all COVID-19 deaths. The focus of prevention and treatment protocols proposed is the preservation of epithelial and endothelial glycocalyx integrity.

Indications of Persistent Glycocalyx Damage in Convalescent COVID-19 Patients: A Prospective Multicenter Study and Hypothesis.

The COVID-19 pandemic is caused by the SARS CoV-2 virus and can lead to severe lung damage and hyperinflammation. In the context of COVID-19 infection, inflammation-induced degradation of the glycocalyx layer in endothelial cells has been demonstrated. Syndecan-1 (SDC-1) is an established parameter for measuring glycocalyx injury. This prospective, multicenter, observational, cross-sectional study analyzed SDC-1 levels in 24 convalescent patients that had been infected with SARS-CoV-2 with mild disease course without need of hospitalization. We included 13 age-matched healthy individuals and 10 age-matched hospitalized COVID-19 patients with acute mild disease course as controls. In convalescent COVID-19 patients, significantly elevated SDC-1 levels were detected after a median of 88 days after symptom onset compared to healthy controls, whereas no difference was found when compared to SDC-1 levels of hospitalized patients undergoing acute disease. This study is the first to demonstrate signs of endothelial damage in non-pre-diseased, convalescent COVID-19 patients after mild disease progression without hospitalization. The data are consistent with studies showing evidence of persistent endothelial damage after severe or critical disease progression. Further work to investigate endothelial damage in convalescent COVID-19 patients should follow.

Kawasaki disease and increased cardiovascular risk: Is there a link to circulating glycocalyx biomarkers?

AIMS: Kawasaki disease (KD) is an acute systemic vasculitis with possible long-term impact of general cardio-vascular health. An endothelial glycocalyx disorder during the disease's acute phase might predispose to long-term vascular anomalies leading to endothelial dysfunction and atherosclerosis. To investigate any association between increased cardiovascular risk and endothelial glycocalyx, we assessed circulating glycocalyx components in patients with a KD history, and analysed their association with acute-phase clinical features and more importantly, with patients' current cardiovascular risk factors. METHODS: This prospective observational cohort study included 51 subjects: 31 patients with a history of KD, and 20 healthy subjects matched for age and sex. We analysed serum syndecan-1 and hyaluronan via ELISA. We assessed features reported during the acute phase of KD such as blood counts, C-reactive protein (CRP) levels and coronary artery aneurysms (CAA), and their current blood pressure and lipid markers in relation to measured glycocalyx components. RESULTS: Our multivariate analysis revealed that hyaluronan and syndecan-1 levels were not associated with KD. However, the latter exhibited a significant association with acute-phase blood count alterations in patients with KD. Furthermore, significant interactions of hyaluronan and syndecan-1 with certain cardiovascular risk factors like blood lipids and blood pressure were only present in KD patients. CONCLUSION: Vasculitis during KD's acute phase might predispose to a long-term endothelial glycocalyx alteration, influenced by other factors having a vascular impact such as blood pressure and circulating lipids. CLINICAL TRIAL REGISTRATION: German Clinical Trials Register on 25th February 2016, DRKS00010071 https://www.drks.de/drks_web/.

COVID-19 generates hyaluronan fragments that directly induce endothelial barrier dysfunction.

Vascular injury has emerged as a complication contributing to morbidity in coronavirus disease 2019 (COVID-19). The glycosaminoglycan hyaluronan (HA) is a major component of the glycocalyx, a protective layer of glycoconjugates that lines the vascular lumen and regulates key endothelial cell functions. During critical illness, as in the case of sepsis, enzymes degrade the glycocalyx, releasing fragments with pathologic activities into circulation and thereby exacerbating disease. Here, we analyzed levels of circulating glycosaminoglycans in 46 patients with COVID-19 ranging from moderate to severe clinical severity and measured activities of corresponding degradative enzymes. This report provides evidence that the glycocalyx becomes significantly damaged in patients with COVID-19 and corresponds with severity of disease. Circulating HA fragments and hyaluronidase, 2 signatures of glycocalyx injury, strongly associate with sequential organ failure assessment scores and with increased inflammatory cytokine levels in patients with COVID-19. Pulmonary microvascular endothelial cells exposed to COVID-19 milieu show dysregulated HA biosynthesis and degradation, leading to production of pathological HA fragments that are released into circulation. Finally, we show that HA fragments present at high levels in COVID-19 patient plasma can directly induce endothelial barrier dysfunction in a ROCK- and CD44-dependent manner, indicating a role for HA in the vascular pathology of COVID-19.

Endothelial glycocalyx shields the interaction of SARS-CoV-2 spike protein with ACE2 receptors.

Endothelial cells (ECs) play a crucial role in the development and propagation of the severe COVID-19 stage as well as multiorgan dysfunction. It remains, however, controversial whether COVID-19-induced endothelial injury is caused directly by the infection of ECs with SARS-CoV-2 or via indirect mechanisms. One of the major concerns is raised by the contradictory data supporting or denying the presence of ACE2, the SARS-CoV-2 binding receptor, on the EC surface. Here, we show that primary human pulmonary artery ECs possess ACE2 capable of interaction with the viral Spike protein (S-protein) and demonstrate the crucial role of the endothelial glycocalyx in the regulation of the S-protein binding to ACE2 on ECs. Using force spectroscopy method, we directly measured ACE2- and glycocalyx-dependent adhesive forces between S-protein and ECs and characterized the nanomechanical parameters of the cells exposed to S-protein. We revealed that the intact glycocalyx strongly binds S-protein but screens its interaction with ACE2. Reduction of glycocalyx layer exposes ACE2 receptors and promotes their interaction with S-protein. These results indicate that the susceptibility of ECs to COVID-19 infection may depend on the glycocalyx condition.

Heparin prevents in vitro glycocalyx shedding induced by plasma from COVID-19 patients.

The severe forms and worsened outcomes of COVID-19 (coronavirus disease 19) are closely associated with hypertension and cardiovascular disease. Endothelial cells express Angiotensin-Converting Enzyme 2 (ACE2), which is the entrance door for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The hallmarks of severe illness caused by SARS-CoV-2 infection are increased levels of IL-6, C-reactive protein, D-dimer, ferritin, neutrophilia and lymphopenia, pulmonary intravascular coagulopathy and microthrombi of alveolar capillaries. The endothelial glycocalyx, a proteoglycan- and glycoprotein-rich layer covering the luminal side of endothelial cells, contributes to vascular homeostasis. It regulates vascular tonus and permeability, prevents thrombosis, and modulates leukocyte adhesion and inflammatory response. We hypothesized that cytokine production and reactive oxygen species (ROS) generation associated with COVID-19 leads to glycocalyx degradation. A cohort of 20 hospitalized patients with a confirmed COVID-19 diagnosis and healthy subjects were enrolled in this study. Mechanisms associated with glycocalyx degradation in COVID-19 were investigated. Increased plasma concentrations of IL-6 and IL1-ß, as well as increased lipid peroxidation and glycocalyx components were detected in plasma from COVID-19 patients compared to plasma from healthy subjects. Plasma from COVID-19 patients induced glycocalyx shedding in cultured human umbilical vein endothelial cells (HUVECs) and disrupted redox balance. Treatment of HUVECs with low molecular weight heparin inhibited the glycocalyx perturbation. In conclusion, plasma from COVID-19 patients promotes glycocalyx shedding and redox imbalance in endothelial cells, and heparin treatment potentially inhibits glycocalyx disruption.

Vascular endothelial injury exacerbates coronavirus disease 2019: The role of endothelial glycocalyx protection.

The potential for a rapid increase in severity is among the most frightening aspects of severe acute respiratory syndrome coronavirus 2 infection. Evidence increasingly suggests that the symptoms of coronavirus disease-2019 (COVID-19)-related acute respiratory distress syndrome (ARDS) differ from those of classic ARDS. Recently, the severity of COVID-19 has been attributed to a systemic, thrombotic, and inflammatory disease that damages not only the lungs but also multiple organs, including the heart, brain, toes, and liver. This systemic form of COVID-19 may be due to inflammation and vascular endothelial cell injury. The vascular endothelial glycocalyx comprises glycoproteins and plays an important role in systemic capillary homeostasis maintenance. The glycocalyx covers the entire vascular endothelium, and its thickness varies among organs. The endothelial glycocalyx is very thin in the pulmonary capillaries, where it is affected by gaseous exchange with the alveoli and the low intravascular pressure in the pulmonary circulation. Despite the clearly important roles of the glycocalyx in vascular endothelial injury, thrombosis, vasculitis, and inflammation, the link between this structure and vascular endothelial cell dysfunction in COVID-19 remains unclear. In this prospective review, we summarize the importance of the glycocalyx and its potential as a therapeutic target in cases of systemic COVID-19.