Epithelial Glycocalyx Research Articles

Matrix metalloproteinases mediate influenza A-associated shedding of the alveolar epithelial glycocalyx.

The alveolar epithelium is protected by a heparan sulfate-rich, glycosaminoglycan layer called the epithelial glycocalyx. It is cleaved in patients with acute respiratory distress syndrome (ARDS) and in murine models of influenza A (IAV) infection, shedding fragments into the airspace from the cell surface. Glycocalyx shedding results in increased permeability of the alveolar-capillary barrier, amplifying acute lung injury. The mechanisms underlying alveolar epithelial glycocalyx shedding in IAV infection are unknown. We hypothesized that induction of host sheddases such as matrix metalloproteinases (MMPs) during IAV infection results in glycocalyx shedding and increased lung injury. We measured glycocalyx shedding and lung injury during IAV infection with and without treatment with the pan-MMP inhibitor Ilomastat (ILO) and in an MMP-7 knock out (MMP-7KO) mouse. C57BL/6 or MMP-7KO male and female mice were given IAV A/PR/8/34 (H1N1) at 30,000 PFU/mouse or PBS intratracheally. For some experiments, C56BL/6 mice were infected in the presence of ILO (100mg/kg) or vehicle given daily by IP injection. Bronchoalveolar lavage (BAL) and lung tissue were collected on day 1, 3, and 7 for analysis of glycocalyx shedding (BAL Syndecan-1) and lung injury (histology, BAL protein, BAL cytokines, BAL immune cell infiltrates, BAL RAGE). Expression and localization of the sheddase MMP-7 and its inhibitor TIMP-1 was examined by RNAScope. For in vitro experiments, MLE-12 mouse lung epithelial cells were cultured and treated with active or heat-inactivated heparinase (2.5 U/mL) prior to infection with IAV (MOI 1) and viral load and MMP-7 and TIMP-1 expression analyzed. IAV infection caused shedding of the epithelial glycocalyx into the BAL. Inhibition of MMPs with ILO reduced glycocalyx shedding by 36% (p = 0.0051) and reduced lung epithelial injury by 40% (p = 0.0404). ILO also reduced viral load by 68% (p = 0.027), despite having no significant effect on lung cytokine production. Both MMP-7 and its inhibitor TIMP-1 were upregulated in IAV infected mice: MMP-7 colocalized with IAV, while TIMP-1 was limited to cells adjacent to infection. However, MMP-7KO mice had similar glycocalyx shedding, epithelial injury, and viral load compared to WT littermates, suggesting redundancy in MMP sheddase function in the lung. In vitro, heparinase treatment before infection led to a 52% increase in viral load (p = 0.0038) without altering MMP-7 or TIMP-1 protein levels. Glycocalyx shedding and MMPs play key roles in IAV-induced epithelial injury, with significant impact on IAV viral load. Further studies are needed to understand which specific MMPs regulate lung epithelial glycocalyx shedding.

Comparative electron microscopic visualization of the lung alveolar epithelial glycocalyx with different staining and labeling methods.

The alveolar surface of the lung is lined by an epithelium consisting of type I (AECI) and type II alveolar epithelial cells (AECII). This epithelium is covered by a liquid alveolar lining layer (ALL). Besides intra-alveolar surfactant, ALL also contains the alveolar epithelial glycocalyx on the apical side of AECI and AECII. To better understand the alveolar epithelial glycocalyx, its ultrastructural visualization by transmission electron microscopy is required. The aim of this study was to systematically re-evaluate routine cytochemical methods for visualization of the alveolar epithelial glycocalyx and specifically its glycan components. For this purpose, we used chemical fixation by vascular perfusion with aldehydes as a common routine approach in mice. After fixation, staining is needed for glycocalyx visualization. Cytochemical staining agents such as alcian blue, ruthenium red, and lanthanum nitrate were compared. In addition, SNL (Sambucus nigra lectin) and UEA1 (Ulex europaeus agglutinin I) were used for sialic acid and fucose-specific labeling. Alcian blue showed the strongest staining, with cloud-like structures, whereas ruthenium red appeared as thread-like structures. On the other hand, lanthanum nitrate did not stain the alveolar epithelial glycocalyx. For specific sialic acid and fucose labeling, both lectins presented a specific signal. In conclusion, these methods can be used routinely for assessing ultrastructural changes of the alveolar epithelial glycocalyx in experimental invivo models under different physiological and pathological conditions. In addition, cytochemical staining by tissue massage and post-embedding lectin labeling after vascular perfusion support 3R (reduction, refinement, replacement) principles of animal welfare.

Prevotella timonensis degrades the vaginal epithelial glycocalyx through high fucosidase and sialidase activities.

Bacterial vaginosis (BV) is a common vaginal infection that affects a significant proportion of women and is associated with reduced fertility and increased risk of secondary infections. Gardnerella vaginalis is the most well-known BV-associated bacterium, but Prevotella species including P. timonensis and P. bivia may also play an important role. We showed that, similar to G. vaginalis, P. timonensis adhered well to the vaginal epithelium, suggesting that both bacteria could be important in the first stage of infection. Compared to the other bacteria, P. timonensis was unique in efficiently removing the protective mucin sugars that cover the vaginal epithelium. These results underscore that vaginal bacteria play different roles in the initiation and development of BV.

Paracellular Delivery of Protein Drugs with Smart EnteroPatho Nanoparticles.

A general platform for the safe and effective oral delivery of biologics would revolutionize the administration of protein-based drugs, improving access for patients and lowering the financial burden on the health-care industry. Because of their dimensions and physiochemical properties, nanomaterials stand as promising vehicles for navigating the complex and challenging environment in the gastrointestinal (GI) tract. Recent developments have led to materials that protect protein drugs from degradation and enable controlled release in the small intestine, the site of absorption for most proteins. Yet, once present in the small intestine, the protein must transit through the secreted mucus and epithelial cells of the intestinal mucosa into systemic circulation, a process that remains a bottleneck for nanomaterial-based delivery. One attractive pathway through the intestinal mucosa is the paracellular route, which avoids cell trafficking and other degradative processes in the interior of cells. Direct flux between cells is regulated by epithelial tight junctions (TJs) that seal the paracellular space and prevent protein flux. Here, we describe a smart nanoparticle system that directly and transiently disrupts TJs for improved protein delivery, an unrealized goal to-date. We take inspiration from enteropathogenic bacteria that adhere to intestinal epithelia and secrete inhibitors that block TJ interactions in the local environment. To mimic these natural mechanisms, we engineer nanoparticles (EnteroPatho NPs) that attach to the epithelial glycocalyx and release TJ modulators in response to the intestinal pH. We show that EnteroPatho NPs lead to TJ disruption and paracellular protein delivery, giving rise to a general platform for oral delivery.

Enzymatic Modulation of the Pulmonary Glycocalyx Enhances Susceptibility to Streptococcus pneumoniae.

The pulmonary epithelial glycocalyx is rich in glycosaminoglycans such as hyaluronan and heparan sulfate. Despite their presence, the importance of these glycosaminoglycans in bacterial lung infections remains elusive. To address this, we intranasally inoculated mice with Streptococcus pneumoniae in the presence or absence of enzymes targeting pulmonary hyaluronan and heparan sulfate, followed by characterization of subsequent disease pathology, pulmonary inflammation, and lung barrier dysfunction. Enzymatic degradation of hyaluronan and heparan sulfate exacerbated pneumonia in mice, as evidenced by increased disease scores and alveolar neutrophil recruitment. However, targeting epithelial hyaluronan in combination with Streptococcus pneumoniae infection further exacerbated systemic disease, indicated by elevated splenic bacterial load and plasma levels of pro-inflammatory cytokines. In contrast, enzymatic cleavage of heparan sulfate resulted in increased bronchoalveolar bacterial burden, lung damage and pulmonary inflammation in mice infected with Streptococcus pneumoniae. Accordingly, heparinase-treated mice also exhibited disrupted lung barrier integrity as evidenced by higher alveolar edema scores and vascular protein leakage into the airways. This finding was corroborated in a human alveolus-on-a-chip platform, confirming that heparinase treatment also disrupts the human lung barrier during Streptococcus pneumoniae infection. Notably, enzymatic pre-treatment with either hyaluronidase or heparinase also rendered human epithelial cells more sensitive to pneumococcal-induced barrier disruption, as determined by transepithelial electrical resistance measurements, consistent with our findings in murine pneumonia. Taken together, these findings demonstrate the importance of intact hyaluronan and heparan sulfate in limiting pneumococci-induced damage, pulmonary inflammation, and epithelial barrier function and integrity.

High-magnitude cyclic stretch induces protein sialylation and RNA de-sialylation at the alveolar epithelial membrane

Sialylated glycans such as sialoglycoproteins, sialoglycolipids and sialoglycoRNA are ubiquitous in the glycocalyx of cells where they play a key role in barrier function, mechanosensation, infection, immune cell traffcking, and inflammation. These functions are particularly relevant at the blood-gas barrier of the lung. The alveolar epithelial glycocalyx is furthermore unique in that it is constantly exposed to cyclic stretch because of normal breathing. Yet, the abundance, regulation and function of sialylated glycans in the alveolar epithelial glycocalyx remains unknown. Here, we focused on the effects of mechanical stretch on the abundance and de novo formation of sialoglyco-proteins and -RNA as major constituents of the alveolar epithelial sialome. Human primary alveolar epithelial cells (hPAEpiC) were exposed to high magnitude cyclic stretch (18% CS) at 0.25 Hz for 24-48 hours to mimic biomechanical forces at the alveolar epithelium during high tidal volume ventilation. Total sialic acids in sialoglycoproteins were labeled with biotin by periodate oxidation and aldehyde ligation (pAL), and total sialic acids in sialoglycoRNA were quantified by 1, 2-diamino-4,5-methylene dioxybenzene ( DMB) assay. De novo formed sialic acids were labeled with biotin using N-azidoacetyl-mannosamine (Ac4ManNAz). Biotinylated siaolglyco-proteins and -RNAs were detected with streptavidin by Western and Northern blots, respectively, and quantified densitometrically. Stretch-dependent changes in the genes regulating sialic acid biosynthesis were assessed by RNA sequencing. High magnitude cyclic stretch increased the sialylation of proteins of different sizes (35-190 kDa) in the hPAEpiC glycocalyx. MGE showed this increase to be attributable to an increased de novo synthesis of sialic acids. On the other hand, high magnitude cyclic stretch reduced the sialylation of hPAEpiC sialoglycoRNA with no alteration in de novo synthesis, suggesting shedding, release or loss of sialoglycoRNA. RNA seq of hPAEpiC revealed that cyclic stretch upregulated the gene GNE (which encodes the enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase), the master regulator of sialic acid biosynthesis. Here, we demonstrate cyclic stretch-induced sialylation of proteins and de-sialylation of RNA in the alveolar epithelial glycocalyx. Given that sialic acids promote the adhesion of both infectious pathogens and immune cells, these effects may contribute to the pathogenesis of ventilator-associated pneumonia and ventilator-induced lung injury. This work is supported by the German Research Foundation, CRC 1449 subproject B01 to MO & WMK. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Multivalent, calcium-independent binding of surfactant protein A and D to sulfated glycosaminoglycans of the alveolar epithelial glycocalyx.

Lung surfactant collectins, surfactant protein A (SP-A) and D (SP-D), are oligomeric C-type lectins involved in lung immunity. Through their carbohydrate recognition domain, they recognize carbohydrates at pathogen surfaces and initiate lung innate immune response. Here, we propose that they may also be able to bind to other carbohydrates present in typical cell surfaces, such as the alveolar epithelial glycocalyx. To test this hypothesis, we analyzed and quantified the binding affinity of SP-A and SP-D to different sugars and glycosaminoglycans (GAGs) by microscale thermophoresis (MST). In addition, by changing the calcium concentration, we aimed to characterize any consequences on the binding behavior. Our results show that both oligomeric proteins bind with high affinity (in nanomolar range) to GAGs, such as hyaluronan (HA), heparan sulfate (HS) and chondroitin sulfate (CS). Binding to HS and CS was calcium-independent, as it was not affected by changing calcium concentration in the buffer. Quantification of GAGs in bronchoalveolar lavage (BAL) fluid from animals deficient in either SP-A or SP-D showed changes in GAG composition, and electron micrographs showed differences in alveolar glycocalyx ultrastructure in vivo. Taken together, SP-A and SP-D bind to model sulfated glycosaminoglycans of the alveolar epithelial glycocalyx in a multivalent and calcium-independent way. These findings provide a potential mechanism for SP-A and SP-D as an integral part of the alveolar epithelial glycocalyx binding and interconnecting free GAGs, proteoglycans, and other glycans in glycoproteins, which may influence glycocalyx composition and structure.NEW & NOTEWORTHY SP-A and SP-D function has been related to innate immunity of the lung based on their binding to sugar residues at pathogen surfaces. However, their function in the healthy alveolus was considered as limited to interaction with surfactant lipids. Here, we demonstrated that these proteins bind to glycosaminoglycans present at typical cell surfaces like the alveolar epithelial glycocalyx. We propose a model where these proteins play an important role in interconnecting alveolar epithelial glycocalyx components.

Bacterial pneumonia-induced shedding of epithelial heparan sulfate inhibits the bactericidal activity of cathelicidin in a murine model.

Bacterial pneumonia is a common clinical syndrome leading to significant morbidity and mortality worldwide. In the current study, we investigate a novel, multidirectional relationship between the pulmonary epithelial glycocalyx and antimicrobial peptides in the setting of methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. Using an in vivo pneumonia model, we demonstrate that highly sulfated heparan sulfate (HS) oligosaccharides are shed into the airspaces in response to MRSA pneumonia. In vitro, these HS oligosaccharides do not directly alter MRSA growth or gene transcription. However, in the presence of an antimicrobial peptide (cathelicidin), increasing concentrations of HS inhibit the bactericidal activity of cathelicidin against MRSA as well as other nosocomial pneumonia pathogens (Klebsiella pneumoniae and Pseudomonas aeruginosa) in a dose-dependent manner. Surface plasmon resonance shows avid binding between HS and cathelicidin with a dissociation constant of 0.13 μM. These findings highlight a complex relationship in which shedding of airspace HS may hamper host defenses against nosocomial infection via neutralization of antimicrobial peptides. These findings may inform future investigation into novel therapeutic targets designed to restore local innate immune function in patients suffering from primary bacterial pneumonia.NEW & NOTEWORTHY Primary Staphylococcus aureus pneumonia causes pulmonary epithelial heparan sulfate (HS) shedding into the airspace. These highly sulfated HS fragments do not alter bacterial growth or transcription, but directly bind with host antimicrobial peptides and inhibit the bactericidal activity of these cationic polypeptides. These findings highlight a complex local interaction between the pulmonary epithelial glycocalyx and antimicrobial peptides in the setting of bacterial pneumonia.

The role of the alveolar epithelial glycocalyx in acute respiratory distress syndrome.

The alveolar epithelial glycocalyx is a dense anionic layer of glycosaminoglycans (GAGs) and proteoglycans that lines the apical surface of the alveolar epithelium. In contrast to the pulmonary endothelial glycocalyx, which has well-established roles in vascular homeostasis and septic organ dysfunction, the alveolar epithelial glycocalyx is less understood. Recent preclinical studies demonstrated that the epithelial glycocalyx is degraded in multiple murine models of acute respiratory distress syndrome (ARDS), particularly those that result from inhaled insults (so-called "direct" lung injury), leading to shedding of GAGs into the alveolar airspaces. Epithelial glycocalyx degradation also occurs in humans with respiratory failure, as quantified by analysis of airspace fluid obtained from ventilator heat moisture exchange (HME) filters. In patients with ARDS, GAG shedding correlates with the severity of hypoxemia and is predictive of the duration of respiratory failure. These effects may be mediated by surfactant dysfunction, as targeted degradation of the epithelial glycocalyx in mice was sufficient to cause increased alveolar surface tension, diffuse microatelectasis, and impaired lung compliance. In this review, we describe the structure of the alveolar epithelial glycocalyx and the mechanisms underlying its degradation during ARDS. We additionally review the current state of knowledge regarding the attributable effect of epithelial glycocalyx degradation in lung injury pathogenesis. Finally, we address glycocalyx degradation as a potential mediator of ARDS heterogeneity, and the subsequent value of point-of-care quantification of GAG shedding to potentially identify patients who are most likely to respond to pharmacological agents aimed at attenuating glycocalyx degradation.

Bidirectional airflow in lung airway-on-a-chip with matrix-derived membrane elicits epithelial glycocalyx formation.

Organ-on-a-chip systems are rapidly advancing as a viable alternative to existing experimental models in respiratory research. To date, however, epithelial cell cultures within lung airway-on-a-chip devices have yet to demonstrate the presence of an epithelial glycocalyx, a thin layer of proteoglycans, glycoproteins, and glycolipids known to play an important role in regulating epithelial function. Here, we demonstrate that an airway-on-a-chip device that incorporates bidirectional flow mimicking breathing cycles in combination with an ultra-thin matrix-derived membrane (UMM) layer can generate a glycocalyx layer comprised of heparan sulfate. Results with this device and airflow system showed dramatic differences of airway epithelial cell viability and expression of tight junctions, cilia, and mucus over a wide range of flow rates when cultured under oscillatory flow. More importantly, for the first time in a microfluidic organ-on-a-chip setting, we achieved the visualization of an airflow-induced epithelial glycocalyx layer. Our experiments highlight the importance of physiological mimicry in developing in vitro models, as bidirectional airflow showed more representative mucociliary differentiation compared to continuous unidirectional airflow. Thus, the lung airway-on-a-chip platform demonstrated in this study holds great potential as a lung epithelial barrier model for studying the mechanisms of various respiratory diseases and for testing the efficacy of therapeutic candidates in the presence of bidirectional airflow and the glycocalyx.

The influenza-injured lung microenvironment promotes MRSA virulence, contributing to severe secondary bacterial pneumonia.

Influenza infection is substantially worsened by the onset of secondary pneumonia caused by bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). The bidirectional interaction between the influenza-injured lung microenvironment and MRSA is poorly understood. By conditioning MRSA exvivo in bronchoalveolar lavage fluid collected from mice at various time points of influenza infection, we found that the influenza-injured lung microenvironment dynamically induces MRSA to increase cytotoxin expression while decreasing metabolic pathways. LukAB, a SaeRS two-component system-dependent cytotoxin, is particularly important to the severity of post-influenza MRSA pneumonia. LukAB's activity is likely shaped by the post-influenza lung microenvironment, as LukAB binds to (and is activated by) heparan sulfate (HS) oligosaccharide sequences shed from the epithelial glycocalyx after influenza. Our findings indicate that post-influenza MRSA pneumonia is shaped by bidirectional host-pathogen interactions: host injury triggers changes in bacterial expression of toxins, the activity of which may be shaped by host-derived HS fragments.

N-Acetylcysteine and Other Sulfur-Donors as a Preventative and Adjunct Therapy for COVID-19.

The airway epithelial glycocalyx plays an important role in preventing severe acute respiratory syndrome coronavirus 2 entry into the epithelial cells, while the endothelial glycocalyx contributes to vascular permeability and tone, as well as modulating immune, inflammatory, and coagulation responses. With ample evidence in the scientific literature that coronavirus disease 2019 (COVID-19) is related to epithelial and endothelial dysfunction, preserving the glycocalyx should be the main focus of any COVID-19 treatment protocol. The most studied functional unit of the glycocalyx is the glycosaminoglycan heparan sulfate, where the degree and position of the sulfate groups determine the biological activity. N-acetylcysteine (NAC) and other sulfur donors contribute to the inorganic sulfate pool, the rate-limiting molecule in sulfation. NAC is not only a precursor to glutathione but also converts to hydrogen sulfide, inorganic sulfate, taurine, Coenzyme A, and albumin. By optimising inorganic sulfate availability, and therefore sulfation, it is proposed that COVID-19 can be prevented or at least most of the symptoms attenuated. A comprehensive COVID-19 treatment protocol is needed to preserve the glycocalyx in both the prevention and treatment of COVID-19. The use of NAC at a dosage of 600 mg bid for the prevention of COVID-19 is proposed, but a higher dosage of NAC (1200 mg bid) should be administered upon the first onset of symptoms. In the severe to critically ill, it is advised that IV NAC should be administered immediately upon hospital admission, and in the late stage of the disease, IV sodium thiosulfate should be considered. Doxycycline as a protease inhibitor will prevent shedding and further degradation of the glycocalyx.

Multi-responsive mesoporous polydopamine composite nanorods cooperate with nano-enzyme and photosensitiser for intensive immunotherapy of bladder cancer.

Bladder cancer is a common malignancy in the urinary system. Defects of drug molecules in bladder during treatment, such as passive diffusion, rapid clearance of periodic urination, poor adhesion and permeation abilities, lead to low delivery efficiency of conventional drugs and high recurrence rate of disease. In this study, we designed multi-responsive mesoporous polydopamine (PDA) composite nanorods cooperating with nano-enzyme and photosensitiser for intensive immunotherapy of bladder cancer. The strongly adhesive mesoporous PDA with wheat germ agglutinin on nanoparticles could specifically adhere to epithelial glycocalyx and made the nanoparticles aggregate in urinary pathways. Meanwhile, 2,3-dimethylmaleic anhydride could be hydrolysed in acidic conditions of tumour microenvironment, giving it a positive charge (charge reversal), which is more amenable to enter cancer cells. Afterwards, manganese dioxide nanorods could catalyse the reaction of excess H2 O2 in tumour microenvironment to generate active oxygen, so as to change the hypoxic environment in tumour, and achieve a pH-responsive for slow release of PD-L1. After the ICG was irradiated by infrared light, a large amount of singlet oxygen was generated, thereby enhancing the therapeutic effect and reducing toxicity in vivo. Besides, mesoporous PDA with indocyanine green photothermal agent could have a local heat up quickly under the near-infrared light to kill cancer cells, thereby enhancing therapeutic efficacy. Accordingly, this mesoporous PDA composite nanorods shed a light on bladder tumour treatment.

Influenza A Virus Causes Shedding of the Alveolar Epithelial Glycocalyx through Activation of Sheddases

ObjectiveThe healthy alveolar epithelium is protected by a heparan sulfate rich, glycosaminoglycan layer called the epithelial glycocalyx. Our group found that the epithelial glycocalyx is shed in patients with acute respiratory distress syndrome (ARDS). In murine models of LPS‐ or bleomycin‐induced acute lung injury, sheddases (membrane‐bound enzymes that cleave extracellular potions of transmembrane proteins) are upregulated and associated with glycocalyx shedding and increased lung permeability. ARDS is commonly caused by viral infections including influenza A (IAV). In murine models, IAV causes massive and persistent glycocalyx shedding into the airspace but the mechanisms by which this occurs are unknown. The objective of this work is to determine the molecular processes underlying IAV‐induced shedding of the glycocalyx.HypothesisWe hypothesize that IAV causes glycocalyx shedding through induction of host sheddases.MethodsWe examined the literature and curated a list of sheddases associated with IAV with potential to cleave the glycocalyx (MMP‐7, ‐2, ‐9 and their inhibitors TIMP‐1 and ‐2). C57BL/6 mice were infected intranasally with A/PR/8/34 (H1N1) at 30,000 PFU/mouse and bronchoalveolar lavage and lung tissue were collected at day 1, 3, and 7 post infection. Sheddase expression was assessed by RT‐qPCR and RNAscope was used to localize lung sheddase expression in infected and uninfected lungs. MLE‐12 mouse lung epithelial cells were infected with viable or heat‐inactivated (56C for 30 min) A/PR/8/34 (H1N1) at a MOI of 1 and sheddase expression measured by RT‐qPCR.ResultsMice infected with IAV develop significant lung inflammation (increased BAL inflammatory cells), lung permeability (increased BAL protein), and increased glycocalyx shedding. MMP‐7 is upregulated in infected vs. uninfected lungs at day 1 and 3 post infection, then returns to baseline levels by day 7. MMP‐7 is only expressed in cells that are directly infected by IAV. Expression of the MMP‐7 inhibitor TIMP‐1 is similar to uninfected lungs on day 1, but increases 50‐fold on day 3. In contrast, MMP‐2 and MMP‐9, as well as their inhibitor TIMP‐2 are not upregulated in the first 7 days after IAV infection. Preliminary studies in lung epithelial cells suggest that heat‐inactivated IAV fails to upregulate MMP‐7.ConclusionsTogether, these data suggest that localized IAV infection increases MMP‐7 in a murine model of IAV infection, but has no effect on several other sheddases. This suggests that MMP‐7 may modulate IAV‐induced glycocalyx shedding. Future studies will explore the mechanisms of IAV induced glycocalyx shedding which could provide molecular targets for clinical intervention in IAV‐ARDS pathogenesis.

Mucins Inhibit Coronavirus Infection in a Glycan-Dependent Manner.

Mucins are a diverse and heterogeneous family of glycoproteins that comprise the bulk of mucus and the epithelial glycocalyx. Mucins are intimately involved in viral transmission. Mucin and virus laden particles can be expelled from the mouth and nose to later infect others. Viruses must also penetrate the mucus layer before cell entry and replication. The role of mucins and their molecular structure have not been well-characterized in coronavirus transmission studies. Laboratory studies predicting high rates of fomite transmission have not translated to real-world infections, and mucins may be one culprit. Here, we probed both surface and direct contact transmission scenarios for their dependence on mucins and their structure. We utilized disease-causing, bovine-derived, human coronavirus OC43. We found that bovine mucins could inhibit the infection of live cells in a concentration- and glycan-dependent manner. The effects were observed in both mock fomite and direct contact transmission experiments and were not dependent upon surface material or time-on-surface. However, the effects were abrogated by removal of the glycans or in a cross-species infection scenario where bovine mucin could not inhibit the infection of a murine coronavirus. Together, our data indicate that the mucin molecular structure plays a complex and important role in host defense.

Alveolar epithelial glycocalyx degradation mediates surfactant dysfunction and contributes to acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a common cause of respiratory failure yet has few pharmacologic therapies, reflecting the mechanistic heterogeneity of lung injury. We hypothesized that damage to the alveolar epithelial glycocalyx, a layer of glycosaminoglycans interposed between the epithelium and surfactant, contributes to lung injury in patients with ARDS. Using mass spectrometry of airspace fluid noninvasively collected from mechanically ventilated patients, we found that airspace glycosaminoglycan shedding (an index of glycocalyx degradation) occurred predominantly in patients with direct lung injury and was associated with duration of mechanical ventilation. Male patients had increased shedding, which correlated with airspace concentrations of matrix metalloproteinases. Selective epithelial glycocalyx degradation in mice was sufficient to induce surfactant dysfunction, a key characteristic of ARDS, leading to microatelectasis and decreased lung compliance. Rapid colorimetric quantification of airspace glycosaminoglycans was feasible and could provide point-of-care prognostic information to clinicians and/or be used for predictive enrichment in clinical trials.

Adhesion and Stiffness of Detached Breast Cancer Cells In Vitro: Co-Treatment with Metformin and 2-Deoxy-d-glucose Induces Changes Related to Increased Metastatic Potential.

Simple SummaryThe process of metastasis is one of the most destructive characteristics of cancer, yet it is still poorly understood. Formation of metastasis comprises several distinct steps: cancer cells first detach from the primary tumor and then enter the bloodstream where they circulate freely in the body and eventually adhere to vessel walls in distant organs where they can form secondary tumors. A recent study discovered that a co-treatment of two common drugs that interfere with cellular metabolism, metformin and 2-deoxy-D-glucose, induced cellular changes resembling those observed in metastasis: the drugs induced detachment of certain breast cancer cells and their proliferation in the floating state. In this study, we investigated if this treatment also induces other changes that are related to metastasis, i.e., if the detached cells are softer and if they are more prone to adhesion than control cells. The results of our in vitro experiments showed that this was indeed the case and thus indicate possible relations between metabolism and metastatic potential. While the results of this study cannot be directly projected to cancers in vivo, they present new observations that can be important for the analysis of cancer cell detachment and anchorage-independent growth.Metastatic cancer cells can overcome detachment-induced cell death and can proliferate in anchorage-independent conditions. A recent study revealed that a co-treatment with two drugs that interfere with cell metabolism, metformin and 2-deoxy-D-glucose, promotes detachment of viable MDA-MB-231 breast cancer cells. In the present study, we analyzed if these detached viable MDA-MB-231 cells also exhibit other features related to cancer metastatic potential, i.e., if they are softer and more prone to adhere to epithelial cells. The cell mechanics of attached cells and floating cells were analyzed by optical tweezers and cell deformability cytometry, respectively. The adhesion was assessed on a confluent monolayer of HUVEC cells, with MDA-MB-231 cells either in static conditions or in a microfluidic flow. Additionally, to test if adhesion was affected by the state of the epithelial glycocalyx, HUVEC cells were treated with neuraminidase and tunicamycin. It was found that the treated MDA-MB-231 cells were more prone to adhere to HUVEC cells and that they were softer than the control, both in the floating state and after re-seeding to a substrate. The changes in the HUVEC glycocalyx, however, did not increase the adhesion potential of MDA-MB-231.

The Epithelial Cell Glycocalyx in Ocular Surface Infection.

The glycocalyx is the main component of the transcellular barrier located at the interface between the ocular surface epithelia and the external environment. This barrier extends up to 500 nm from the plasma membrane and projects into the tear fluid bathing the surface of the eye. Under homeostatic conditions, defense molecules in the glycocalyx, such as transmembrane mucins, resist infection. However, many pathogenic microorganisms have evolved to exploit components of the glycocalyx in order to gain access to epithelial cells and consequently exert deleterious effects. This manuscript reviews the implications of the ocular surface epithelial glycocalyx to bacterial, viral, fungal and parasitic infection. Moreover, it presents some ongoing controversies surrounding the functional relevance of the epithelial glycocalyx to ocular infectious disease.

Experimental Degradation of the Alveolar Epithelial Glycocalyx Decreases Lung Compliance in Young and Old Mice, Potentially by Inducing Surfactant Dysfunction

Background The alveolar airspace is lined by a heparan sulfate (HS)-enriched epithelial glycocalyx. We previously demonstrated that direct lung injury (i.e., caused by inhalational insults) causes shedding of epithelial HS into the airspaces of mice and humans. However, the homeostatic function of epithelial surface HS is uncertain. We hypothesized that during lung homeostasis, epithelial HS contributes to the aqueous hypophase upon which surfactant is distributed. As such, experimental degradation of epithelial HS should lead to alveolar collapse and loss of lung compliance, key characteristics of ARDS pathophysiology. Methods and results: We treated young (8-10 week) and old (>22 month) C57BL/6 mice with 15 units intratracheal heparinase I/III (Hep I/III), a HS-specific glucuronidase that specifically degrades epithelial surface HS. Control mice received equal concentrations of heat-inactivated Hep I/III (HI-Hep I/III). 24 hours later, Hep I/III-treated mice demonstrated loss of inspiratory capacity (0.6114 ml vs. 0.7158 ml, n = 10 per group; p < 0.05) and loss of lung compliance (0.049 ml/cm H2O vs. 0.059 ml/cm H2O, n = 10, p < 0.05); similar results were seen in aged mice (1.061 ml vs. 1.170 ml; 0.082 ml/cm H2O vs 0.093 ml/cm H2O; n =3-4, p < 0.05 for both). Surprisingly, this rapid loss of compliance occurred in the absence of lung edema (as measured by wet/dry ratio and histology), fibrosis (histology), or inflammation (RNA sequencing, BAL cell count and differential). Micro-CT scans of the lungs of Hep I/III-treated young mice at increasing PEEP confirmed a loss of lung compliance and an increase in radiographic lung density (Hounsfield units). Preliminary lung stereology demonstrated the presence of microatelectasis, suggesting that loss of airspace HS could induce surfactant dysfunction. As BAL phospholipid analyses showed no differences in phospholipid species between Hep I/III- and HI-Hep I/III young mice, loss of airspace HS may cause functional impairments in surfactant, as opposed to alterations in surfactant expression. Conclusions Our findings suggest that loss of alveolar epithelial HS disrupts surfactant function, potentially via disrupting the epithelial aqueous hypophase. Reconstitution of epithelial glycocalyx integrity after ARDS would therefore be essential to lung repair and the subsequent return to homeostasis.

Vagus Nerve Stimulation Protects Enterocyte Glycocalyx After Hemorrhagic Shock Via the Cholinergic Anti-Inflammatory Pathway.

ABSTRACTIntroduction:Electrical vagal nerve stimulation is known to decrease gut permeability and alleviate gut injury caused by traumatic hemorrhagic shock. However, the specific mechanism of action remains unclear. Glycocalyx, located on the surface of the intestinal epithelium, is associated with the buildup of the intestinal barrier. Therefore, the goal of our study was to explore whether vagal nerve stimulation affects enterocyte glycocalyx, gut permeability, gut injury, and remote lung injury.Materials and methods:Male Sprague Dawley rats were anesthetized and their cervical nerves were exposed. The rats underwent traumatic hemorrhagic shock (with maintenance of mean arterial pressure of 30–35 mmHg for 60 min) with fluid resuscitation. Vagal nerve stimulation was added to two cohorts of animals before fluid resuscitation, and one of them was injected with methyllycaconitine to block the cholinergic anti-inflammatory pathway. Intestinal epithelial glycocalyx was detected using immunofluorescence. Intestinal permeability, the degree of gut and lung injury, and inflammation factors were also assessed.Results:Vagal nerve stimulation alleviated the damage to the intestinal epithelial glycocalyx and decreased intestinal permeability by 43% compared with the shock/resuscitation phase (P < 0.05). Methyllycaconitine partly eliminated the effects of vagal nerve stimulation on the intestinal epithelial glycocalyx (P < 0.05). Vagal nerve stimulation protected against traumatic hemorrhagic shock/fluid resuscitation-induced gut and lung injury, and some inflammatory factor levels in the gut and lung tissue were downregulated after vagal nerve stimulation (P < 0.05).Conclusions:Vagal nerve stimulation could relieve traumatic hemorrhagic shock/fluid resuscitation-induced intestinal epithelial glycocalyx damage via the cholinergic anti-inflammatory pathway.