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.

Membrane-tethered mucin-like polypeptides sterically inhibit binding and slow fusion kinetics of influenza A virus.

The mechanism(s) by which cell-tethered mucins modulate infection by influenza A viruses (IAVs) remain an open question. Mucins form both a protective barrier that can block virus binding and recruit IAVs to bind cells via the sialic acids of cell-tethered mucins. To elucidate the molecular role of mucins in flu pathogenesis, we constructed a synthetic glycocalyx to investigate membrane-tethered mucins in the context of IAV binding and fusion. We designed and synthesized lipid-tethered glycopolypeptide mimics of mucins and added them to lipid bilayers, allowing chemical control of length, glycosylation, and surface density of a model glycocalyx. We observed that the mucin mimics undergo a conformational change at high surface densities from a compact to an extended architecture. At high surface densities, asialo mucin mimics inhibited IAV binding to underlying glycolipid receptors, and this density correlated to the mucin mimic's conformational transition. Using a single virus fusion assay, we observed that while fusion of virions bound to vesicles coated with sialylated mucin mimics was possible, the kinetics of fusion was slowed in a mucin density-dependent manner. These data provide a molecular model for a protective mechanism by mucins in IAV infection, and therefore this synthetic glycocalyx provides a useful reductionist model for studying the complex interface of host-pathogen interactions.

Macrophage migration inhibitory factor is critical for dengue NS1-induced endothelial glycocalyx degradation and hyperpermeability.

Vascular leakage is one of the salient characteristics of severe dengue. Nonstructural protein 1 (NS1) of dengue virus (DENV) can stimulate endothelial cells to secrete endothelial hyperpermeability factor, macrophage migration inhibitory factor (MIF), and the glycocalyx degradation factor heparanase 1 (HPA-1). However, it is unclear whether MIF is directly involved in NS1-induced glycocalyx degradation. In this study, we observed that among NS1, MIF and glycocalyx degradation-related molecules, the HPA-1, metalloproteinase 9 (MMP-9) and syndecan 1 (CD138) serum levels were all increased in dengue patients, and only NS1 and MIF showed a positive correlation with the CD138 level in severe patients. To further characterize and clarify the relationship between MIF and CD138, we used recombinant NS1 to stimulate human cells in vitro and challenge mice in vivo. Our tabulated results suggested that NS1 stimulation could induce human endothelial cells to secrete HPA-1 and immune cells to secrete MMP-9, resulting in endothelial glycocalyx degradation and hyperpermeability. Moreover, HPA-1, MMP-9, and CD138 secretion after NS1 stimulation was blocked by MIF inhibitors or antibodies both in vitro and in mice. Taken together, these results suggest that MIF directly engages in dengue NS1-induced glycocalyx degradation and that targeting MIF may represent a possible therapeutic approach for preventing dengue-induced vascular leakage.

Dengue Virus NS1 Disrupts the Endothelial Glycocalyx, Leading to Hyperpermeability.

Dengue is the most prevalent arboviral disease in humans and a major public health problem worldwide. Systemic plasma leakage, leading to hypovolemic shock and potentially fatal complications, is a critical determinant of dengue severity. Recently, we and others described a novel pathogenic effect of secreted dengue virus (DENV) non-structural protein 1 (NS1) in triggering hyperpermeability of human endothelial cells in vitro and systemic vascular leakage in vivo. NS1 was shown to activate toll-like receptor 4 signaling in primary human myeloid cells, leading to secretion of pro-inflammatory cytokines and vascular leakage. However, distinct endothelial cell-intrinsic mechanisms of NS1-induced hyperpermeability remained to be defined. The endothelial glycocalyx layer (EGL) is a network of membrane-bound proteoglycans and glycoproteins lining the vascular endothelium that plays a key role in regulating endothelial barrier function. Here, we demonstrate that DENV NS1 disrupts the EGL on human pulmonary microvascular endothelial cells, inducing degradation of sialic acid and shedding of heparan sulfate proteoglycans. This effect is mediated by NS1-induced expression of sialidases and heparanase, respectively. NS1 also activates cathepsin L, a lysosomal cysteine proteinase, in endothelial cells, which activates heparanase via enzymatic cleavage. Specific inhibitors of sialidases, heparanase, and cathepsin L prevent DENV NS1-induced EGL disruption and endothelial hyperpermeability. All of these effects are specific to NS1 from DENV1-4 and are not induced by NS1 from West Nile virus, a related flavivirus. Together, our data suggest an important role for EGL disruption in DENV NS1-mediated endothelial dysfunction during severe dengue disease.

Association between plasma glycocalyx component levels and poor prognosis in severe influenza type A (H1N1).

Influenza A virus infection causes a series of diseases, but the factors associated with disease severity are not fully understood. Disruption of the endothelial glycocalyx contributes to acute lung injury in sepsis, but has not been well studied in H1N1 influenza. We aim to determine whether the plasma glycocalyx components levels are predictive of disease severity in H1N1 influenza. This prospective observational study included 53 patients with influenza A (H1N1) during the influenza season, and 30 healthy controls in our hospital. Patients were grouped by severity and survival. We collected clinical data and blood samples at admission. Inflammatory factors (tumor necrosis factor-α, interleukin-6, interleukin-10) and endothelial glycocalyx components (syndecan-1, hyaluronan, heparan sulfate) were measured. The plasma levels of syndecan-1, hyaluronan, and heparan sulfate were significantly higher in patients with severe influenza A (H1N1) than in mild cases. Syndecan-1 and hyaluronan were positively correlated with disease severity, which was indicated by the APACHE II and SOFA scores and lactate levels, and negatively correlated with albumin levels. At a cutoff point ≥ 173.9 ng/mL, syndecan-1 had a 81.3% sensitivity and 70.3% specificity for predicting of 28-day mortality. Kaplan-Meier analysis demonstrated a strong association between syndecan-1 levels and 28-day mortality (log-rank 11.04, P = 0.001). Elevated plasma levels of syndecan-1 has a potential role in systemic organ dysfunction and may be indicative of disease severity in patients with influenza A (H1N1).

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.

Endothelial glycocalyx damage as a systemic inflammatory microvascular endotheliopathy in COVID-19.

In atherosclerosis patients, vascular endothelial dysfunction is commonly observed alongside damage of the vascular endothelial glycocalyx, an extracellular matrix bound to and encapsulating the endothelial cells lining the blood vessel wall. Although atherosclerotic risk factors have been reported in severe patients with coronavirus disease 2019 (COVID-19), the exact mechanisms are unclear. The mortality associated with the COVID-19 outbreak is increased by comorbidities, including hypertension, diabetes, obesity, chronic obstructive pulmonary disease (COPD), and cardiovascular disease. Besides, older individuals and smokers have significantly worse outcomes. Interestingly, these comorbidities and risk factors are consistent with the pathophysiology that causes vascular endothelial glycocalyx damage. Moreover, vascular glycocalyx dysfunction causes microvascular leakage, which results in interstitial pulmonary abnormal shadows (multiple patchy shadows with a ground glass inter-pneumonic appearance). This is frequently followed by severe acute respiratory distress syndrome (ARDS), closely related to coagulo-fibrinolytic changes contributing to disseminated intravascular coagulation (DIC) and Kawasaki disease shock syndrome, as well as inducing activation of the coagulation cascade, leading to thromboembolism and multiple organ failure. Notably, SARS-CoV-2, the causative virus of COVID-19, binds to ACE2, which is abundantly present not only in human epithelia of the lung and the small intestine, but also in vascular endothelial cells and arterial smooth muscle cells. Moreover, COVID-19 can induce severe septic shock, and sepsis can easily lead to systemic degradation of the vascular endothelial glycocalyx. In the current review, we propose new concepts and therapeutic goals for COVID-19-related vascular endothelial glycocalyx damage, based on previous vascular endothelial medicine research.

Flavivirus NS1 Triggers Tissue-Specific Vascular Endothelial Dysfunction Reflecting Disease Tropism.

Flaviviruses cause systemic or neurotropic-encephalitic pathology in humans. The flavivirus nonstructural protein 1 (NS1) is a secreted glycoprotein involved in viral replication, immune evasion, and vascular leakage during dengue virus infection. However, the contribution of secreted NS1 from related flaviviruses to viral pathogenesis remains unknown. Here, we demonstrate that NS1 from dengue, Zika, West Nile, Japanese encephalitis, and yellow fever viruses selectively binds to and alters permeability of human endothelial cells from lung, dermis, umbilical vein, brain, and liver in vitro and causes tissue-specific vascular leakage in mice, reflecting the pathophysiology of each flavivirus. Mechanistically, each flavivirus NS1 leads to differential disruption of endothelial glycocalyx components, resulting in endothelial hyperpermeability. Our findings reveal the capacity of a secreted viral protein to modulate endothelial barrier function in a tissue-specific manner both in vitro and in vivo, potentially influencing virus dissemination and pathogenesis and providing targets for antiviral therapies and vaccine development.