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Brain metastatic breast cancer is particularly lethal largely due to therapeutic resistance. Almost half of the patients with metastatic HER2-positive breast cancer develop brain metastases, representing a major clinical challenge. We previously described that cancer-associated fibroblasts are an important source of resistance in primary tumors. Here, we report that breast cancer brain metastasis stromal cell interactions in 3D cocultures induce therapeutic resistance to HER2-targeting agents, particularly to the small molecule inhibitor of HER2/EGFR neratinib. We investigated the underlying mechanisms using a synthetic Notch reporter system enabling the sorting of cancer cells that directly interact with stromal cells. We identified mucins and bulky glycoprotein synthesis as top-up-regulated genes and pathways by comparing the gene expression and chromatin profiles of stroma-contact and no-contact cancer cells before and after neratinib treatment. Glycoprotein gene signatures were also enriched in human brain metastases compared to primary tumors. We confirmed increased glycocalyx surrounding cocultures by immunofluorescence and showed that mucinase treatment increased sensitivity to neratinib by enabling a more efficient inhibition of EGFR/HER2 signaling in cancer cells. Overexpression of truncated MUC1 lacking the intracellular domain as a model of increased glycocalyx-induced resistance to neratinib both in cell culture and in experimental brain metastases in immunodeficient mice. Our results highlight the importance of glycoproteins as a resistance mechanism to HER2-targeting therapies in breast cancer brain metastases.

Sepsis is a condition with high morbidity and mortality. Prompt recognition and initiation of treatment is essential. Despite forming an integral part of sepsis management, fluid resuscitation may also lead to volume overload, which in turn is associated with increased mortality. The optimal fluid strategy in sepsis resuscitation is yet to be defined. Hyaluronan, an endogenous glycosaminoglycan with high affinity to water is an important constituent of the endothelial glycocalyx. We hypothesized that exogenously administered hyaluronan would counteract intravascular volume depletion and contribute to endothelial glycocalyx integrity in a fluid restrictive model of peritonitis. In a prospective, blinded model of porcine peritonitis sepsis, we randomized animals to intervention with hyaluronan (n = 8) or 0.9% saline (n = 8). The animals received an infusion of 0.1% hyaluronan 6 ml/kg/h, or the same volume of saline, during the first 2 h of peritonitis. Stroke volume variation and hemoconcentration were comparable in the two groups throughout the experiment. Cardiac output was higher in the intervention group during the infusion of hyaluronan (3.2 ± 0.5 l/min in intervention group vs 2.7 ± 0.2 l/min in the control group) (p = 0.039). The increase in lactate was more pronounced in the intervention group (3.2 ± 1.0 mmol/l in the intervention group and 1.7 ± 0.7 mmol/l in the control group) at the end of the experiment (p < 0.001). Concentrations of surrogate markers of glycocalyx damage; syndecan 1 (0.6 ± 0.2 ng/ml vs 0.5 ± 0.2 ng/ml, p = 0.292), heparan sulphate (1.23 ± 0.2 vs 1.4 ± 0.3 ng/ml, p = 0.211) and vascular adhesion protein 1 (7.0 ± 4.1 vs 8.2 ± 2.3 ng/ml, p = 0.492) were comparable in the two groups at the end of the experiment. In conclusion, hyaluronan did not counteract intravascular volume depletion in early peritonitis sepsis. However, this finding is hampered by the short observation period and a beneficial effect of HMW-HA in peritonitis sepsis cann, Title in the list of papers of Annelie Barrueta Tenhunen's thesis: Fluid restrictive resuscitation with high molecular weight hyaluronan infusion in early peritonitis-sepsis

Sepsis is a condition with high morbidity and mortality. Prompt recognition and initiation of treatment is essential. Despite forming an integral part of sepsis management, fluid resuscitation may also lead to volume overload, which in turn is associated with increased mortality. The optimal fluid strategy in sepsis resuscitation is yet to be defined. Hyaluronan, an endogenous glycosaminoglycan with high affinity to water is an important constituent of the endothelial glycocalyx. We hypothesized that exogenously administered hyaluronan would counteract intravascular volume depletion and contribute to endothelial glycocalyx integrity in a fluid restrictive model of peritonitis. In a prospective, blinded model of porcine peritonitis sepsis, we randomized animals to intervention with hyaluronan (n = 8) or 0.9% saline (n = 8). The animals received an infusion of 0.1% hyaluronan 6 ml/kg/h, or the same volume of saline, during the first 2 h of peritonitis. Stroke volume variation and hemoconcentration were comparable in the two groups throughout the experiment. Cardiac output was higher in the intervention group during the infusion of hyaluronan (3.2 ± 0.5 l/min in intervention group vs 2.7 ± 0.2 l/min in the control group) (p = 0.039). The increase in lactate was more pronounced in the intervention group (3.2 ± 1.0 mmol/l in the intervention group and 1.7 ± 0.7 mmol/l in the control group) at the end of the experiment (p < 0.001). Concentrations of surrogate markers of glycocalyx damage; syndecan 1 (0.6 ± 0.2 ng/ml vs 0.5 ± 0.2 ng/ml, p = 0.292), heparan sulphate (1.23 ± 0.2 vs 1.4 ± 0.3 ng/ml, p = 0.211) and vascular adhesion protein 1 (7.0 ± 4.1 vs 8.2 ± 2.3 ng/ml, p = 0.492) were comparable in the two groups at the end of the experiment. In conclusion, hyaluronan did not counteract intravascular volume depletion in early peritonitis sepsis. However, this finding is hampered by the short observation period and a beneficial effect of HMW-HA in peritonitis sepsis cann, Title in the list of papers of Annelie Barrueta Tenhunen's thesis: Fluid restrictive resuscitation with high molecular weight hyaluronan infusion in early peritonitis-sepsis

Plasma polymers (PPs) can easily modify material surfaces to improve their bio-applicability due to match-made surface-free energy and functionality. However, cell adhesion to PPs typically composed of various functional groups has not yet been fully understood. We explain the origin of strong resistance to trypsin treatment previously noted for nonendothelial cells on amine PPs. It is caused mainly by nonspecific adhesion of negatively charged parts of transmembrane proteins to the positively charged amine PP surface, enabled by thin glycocalyx. However, endothelial cells are bound primarily by their thick, negatively charged glycocalyx and sporadically by integrins in kinetic traps, both cleaved by trypsin. Cell scratching by atomic force microscopy tip confirmed the correlation of trypsin resistance to the strength of cell adhesion.

Lipoprotein lipase (LPL), the enzyme that carries out the lipolytic processing of triglyceride-rich lipoproteins (TRLs), is synthesized by adipocytes and myocytes and secreted into the interstitial spaces. The LPL is then bound by GPIHBP1, a GPI-anchored protein of endothelial cells (ECs), and transported across ECs to the capillary lumen. The assumption has been that the LPL that is moved into capillaries remains attached to GPIHBP1 and that GPIHBP1 serves as a platform for TRL processing. In the current studies, we examined the validity of that assumption. We found that an LPL-specific monoclonal antibody (mAb), 88B8, which lacks the ability to detect GPIHBP1-bound LPL, binds avidly to LPL within capillaries. We further demonstrated, by confocal microscopy, immunogold electron microscopy, and nanoscale secondary ion mass spectrometry analyses, that the LPL detected by mAb 88B8 is located within the EC glycocalyx, distant from the GPIHBP1 on the EC plasma membrane. The LPL within the glycocalyx mediates the margination of TRLs along capillaries and is active in TRL processing, resulting in the delivery of lipoprotein-derived lipids to immediately adjacent parenchymal cells. Thus, the LPL that GPIHBP1 transports into capillaries can detach and move into the EC glycocalyx, where it functions in the intravascular processing of TRLs.

Clathrin-mediated endocytosis is essential for the removal of transmembrane proteins from the plasma membrane in all eukaryotic cells. Many transmembrane proteins are glycosylated. These proteins collectively comprise the glycocalyx, a sugar-rich layer at the cell surface, which is responsible for intercellular adhesion and recognition. Previous work has suggested that glycosylation of transmembrane proteins reduces their removal from the plasma membrane by endocytosis. However, the mechanism responsible for this effect remains unknown. To study the impact of glycosylation on endocytosis, we replaced the ectodomain of the transferrin receptor, a well-studied transmembrane protein that undergoes clathrin-mediated endocytosis, with the ectodomain of MUC1, which is highly glycosylated. When we expressed this transmembrane fusion protein in mammalian epithelial cells, we found that its recruitment to endocytic structures was substantially reduced in comparison to a version of the protein that lacked the MUC1 ectodomain. This reduction could not be explained by a loss of mobility on the cell surface or changes in endocytic dynamics. Instead, we found that the bulky MUC1 ectodomain presented a steric barrier to endocytosis. Specifically, the peptide backbone of the ectodomain and its glycosylation each made steric contributions, which drove comparable reductions in endocytosis. These results suggest that glycosylation constitutes a biophysical signal for retention of transmembrane proteins at the plasma membrane. This mechanism could be modulated in multiple disease states that exploit the glycocalyx, from cancer to atherosclerosis.

Sepsis is a condition with high morbidity and mortality. Prompt recognition and initiation of treatment is essential. Despite forming an integral part of sepsis management, fluid resuscitation may also lead to volume overload, which in turn is associated with increased mortality. The optimal fluid strategy in sepsis resuscitation is yet to be defined. Hyaluronan, an endogenous glycosaminoglycan with high affinity to water is an important constituent of the endothelial glycocalyx. We hypothesized that exogenously administered hyaluronan would counteract intravascular volume depletion and contribute to endothelial glycocalyx integrity in a fluid restrictive model of peritonitis. In a prospective, blinded model of porcine peritonitis sepsis, we randomized animals to intervention with hyaluronan (n = 8) or 0.9% saline (n = 8). The animals received an infusion of 0.1% hyaluronan 6 ml/kg/h, or the same volume of saline, during the first 2 h of peritonitis. Stroke volume variation and hemoconcentration were comparable in the two groups throughout the experiment. Cardiac output was higher in the intervention group during the infusion of hyaluronan (3.2 ± 0.5 l/min in intervention group vs 2.7 ± 0.2 l/min in the control group) (p = 0.039). The increase in lactate was more pronounced in the intervention group (3.2 ± 1.0 mmol/l in the intervention group and 1.7 ± 0.7 mmol/l in the control group) at the end of the experiment (p < 0.001). Concentrations of surrogate markers of glycocalyx damage; syndecan 1 (0.6 ± 0.2 ng/ml vs 0.5 ± 0.2 ng/ml, p = 0.292), heparan sulphate (1.23 ± 0.2 vs 1.4 ± 0.3 ng/ml, p = 0.211) and vascular adhesion protein 1 (7.0 ± 4.1 vs 8.2 ± 2.3 ng/ml, p = 0.492) were comparable in the two groups at the end of the experiment. In conclusion, hyaluronan did not counteract intravascular volume depletion in early peritonitis sepsis. However, this finding is hampered by the short observation period and a beneficial effect of HMW-HA in peritonitis sepsis cann, Title in the list of papers of Annelie Barrueta Tenhunen's thesis: Fluid restrictive resuscitation with high molecular weight hyaluronan infusion in early peritonitis-sepsis

Sepsis is a condition with high morbidity and mortality. Prompt recognition and initiation of treatment is essential. Despite forming an integral part of sepsis management, fluid resuscitation may also lead to volume overload, which in turn is associated with increased mortality. The optimal fluid strategy in sepsis resuscitation is yet to be defined. Hyaluronan, an endogenous glycosaminoglycan with high affinity to water is an important constituent of the endothelial glycocalyx. We hypothesized that exogenously administered hyaluronan would counteract intravascular volume depletion and contribute to endothelial glycocalyx integrity in a fluid restrictive model of peritonitis. In a prospective, blinded model of porcine peritonitis sepsis, we randomized animals to intervention with hyaluronan (n = 8) or 0.9% saline (n = 8). The animals received an infusion of 0.1% hyaluronan 6 ml/kg/h, or the same volume of saline, during the first 2 h of peritonitis. Stroke volume variation and hemoconcentration were comparable in the two groups throughout the experiment. Cardiac output was higher in the intervention group during the infusion of hyaluronan (3.2 ± 0.5 l/min in intervention group vs 2.7 ± 0.2 l/min in the control group) (p = 0.039). The increase in lactate was more pronounced in the intervention group (3.2 ± 1.0 mmol/l in the intervention group and 1.7 ± 0.7 mmol/l in the control group) at the end of the experiment (p < 0.001). Concentrations of surrogate markers of glycocalyx damage; syndecan 1 (0.6 ± 0.2 ng/ml vs 0.5 ± 0.2 ng/ml, p = 0.292), heparan sulphate (1.23 ± 0.2 vs 1.4 ± 0.3 ng/ml, p = 0.211) and vascular adhesion protein 1 (7.0 ± 4.1 vs 8.2 ± 2.3 ng/ml, p = 0.492) were comparable in the two groups at the end of the experiment. In conclusion, hyaluronan did not counteract intravascular volume depletion in early peritonitis sepsis. However, this finding is hampered by the short observation period and a beneficial effect of HMW-HA in peritonitis sepsis cann

Plasma polymers (PPs) can easily modify material surfaces to improve their bio-applicability due to match-made surface-free energy and functionality. However, cell adhesion to PPs typically composed of various functional groups has not yet been fully understood. We explain the origin of strong resistance to trypsin treatment previously noted for nonendothelial cells on amine PPs. It is caused mainly by nonspecific adhesion of negatively charged parts of transmembrane proteins to the positively charged amine PP surface, enabled by thin glycocalyx. However, endothelial cells are bound primarily by their thick, negatively charged glycocalyx and sporadically by integrins in kinetic traps, both cleaved by trypsin. Cell scratching by atomic force microscopy tip confirmed the correlation of trypsin resistance to the strength of cell adhesion.

Plasma polymers (PPs) can easily modify material surfaces to improve their bio-applicability due to match-made surface-free energy and functionality. However, cell adhesion to PPs typically composed of various functional groups has not yet been fully understood. We explain the origin of strong resistance to trypsin treatment previously noted for nonendothelial cells on amine PPs. It is caused mainly by nonspecific adhesion of negatively charged parts of transmembrane proteins to the positively charged amine PP surface, enabled by thin glycocalyx. However, endothelial cells are bound primarily by their thick, negatively charged glycocalyx and sporadically by integrins in kinetic traps, both cleaved by trypsin. Cell scratching by atomic force microscopy tip confirmed the correlation of trypsin resistance to the strength of cell adhesion.

Adipose tissue plays a crucial role in maintaining metabolic homeostasis by storing lipids and glucose from circulation as intracellular fat. As peripheral tissues like adipose tissue become insulin resistant, decompensation of blood glucose levels occurs causing type 2 diabetes (T2D). Currently, modulating the glycocalyx, a layer of cell-surface glycans, is an underexplored pharmacological treatment strategy to improve glucose homeostasis in T2D patients. Here, we show a novel role for cell-surface heparan sulfate (HS) in establishing glucose uptake capacity and metabolic utilization in differentiated adipocytes. Using a combination of chemical and genetic interventions, we identified that HS modulates this metabolic phenotype by attenuating levels of Wnt signaling during adipogenesis. By engineering, the glycocalyx of pre-adipocytes with exogenous synthetic HS mimetics, we were able to enhance glucose clearance capacity after differentiation through modulation of Wnt ligand availability. These findings establish the cellular glycocalyx as a possible new target for therapeutic intervention in T2D patients by enhancing glucose clearance capacity independent of insulin secretion.

Cells mediate interactions with the extracellular environment through a crowded assembly of transmembrane proteins, glycoproteins and glycolipids on their plasma membrane. The extent to which surface crowding modulates the biophysical interactions of ligands, receptors, and other macromolecules is poorly understood due to the lack of methods to quantify surface crowding on native cell membranes. In this work, we demonstrate that physical crowding on reconstituted membranes and live cell surfaces attenuates the effective binding affinity of macromolecules such as IgG antibodies in a surface crowding-dependent manner. We combine experiment and simulation to design a crowding sensor based on this principle that provides a quantitative readout of cell surface crowding. Our measurements reveal that surface crowding decreases IgG antibody binding by 2 to 20 fold in live cells compared to a bare membrane surface. Our sensors show that sialic acid, a negatively charged monosaccharide, contributes disproportionately to red blood cell surface crowding via electrostatic repulsion, despite occupying only ~1% of the total cell membrane by mass. We also observe significant differences in surface crowding for different cell types and find that expression of single oncogenes can both increase and decrease crowding, suggesting that surface crowding may be an indicator of both cell type and state. Our high-throughput, single-cell measurement of cell surface crowding may be combined with functional assays to enable further biophysical dissection of the cell surfaceome.

Sepsis is a condition with high morbidity and mortality. Prompt recognition and initiation of treatment is essential. Despite forming an integral part of sepsis management, fluid resuscitation may also lead to volume overload, which in turn is associated with increased mortality. The optimal fluid strategy in sepsis resuscitation is yet to be defined. Hyaluronan, an endogenous glycosaminoglycan with high affinity to water is an important constituent of the endothelial glycocalyx. We hypothesized that exogenously administered hyaluronan would counteract intravascular volume depletion and contribute to endothelial glycocalyx integrity in a fluid restrictive model of peritonitis. In a prospective, blinded model of porcine peritonitis sepsis, we randomized animals to intervention with hyaluronan (n = 8) or 0.9% saline (n = 8). The animals received an infusion of 0.1% hyaluronan 6 ml/kg/h, or the same volume of saline, during the first 2 h of peritonitis. Stroke volume variation and hemoconcentration were comparable in the two groups throughout the experiment. Cardiac output was higher in the intervention group during the infusion of hyaluronan (3.2 ± 0.5 l/min in intervention group vs 2.7 ± 0.2 l/min in the control group) (p = 0.039). The increase in lactate was more pronounced in the intervention group (3.2 ± 1.0 mmol/l in the intervention group and 1.7 ± 0.7 mmol/l in the control group) at the end of the experiment (p < 0.001). Concentrations of surrogate markers of glycocalyx damage; syndecan 1 (0.6 ± 0.2 ng/ml vs 0.5 ± 0.2 ng/ml, p = 0.292), heparan sulphate (1.23 ± 0.2 vs 1.4 ± 0.3 ng/ml, p = 0.211) and vascular adhesion protein 1 (7.0 ± 4.1 vs 8.2 ± 2.3 ng/ml, p = 0.492) were comparable in the two groups at the end of the experiment. In conclusion, hyaluronan did not counteract intravascular volume depletion in early peritonitis sepsis. However, this finding is hampered by the short observation period and a beneficial effect of HMW-HA in peritonitis sepsis cann, Title in the list of papers of Annelie Barrueta Tenhunen's thesis: Fluid restrictive resuscitation with high molecular weight hyaluronan infusion in early peritonitis-sepsis

Plasma polymers (PPs) can easily modify material surfaces to improve their bio-applicability due to match-made surface-free energy and functionality. However, cell adhesion to PPs typically composed of various functional groups has not yet been fully understood. We explain the origin of strong resistance to trypsin treatment previously noted for nonendothelial cells on amine PPs. It is caused mainly by nonspecific adhesion of negatively charged parts of transmembrane proteins to the positively charged amine PP surface, enabled by thin glycocalyx. However, endothelial cells are bound primarily by their thick, negatively charged glycocalyx and sporadically by integrins in kinetic traps, both cleaved by trypsin. Cell scratching by atomic force microscopy tip confirmed the correlation of trypsin resistance to the strength of cell adhesion.

A multi-glycomic method for characterizing the glycocalyx was employed to identify the difference between 2-dimensional (2D) and 3-dimensional (3D) culture models with two human colorectal cancer cell lines, HCT116 and HT29. 3D cell cultures are considered more representative of cancer due to their ability to mimic the microenvironment found in tumors. For this reason, they have become an important tool in cancer research. Cell-cell interactions increase in 3D models compared to 2D, indeed significant glycomic changes were observed for each cell line. Analyses included the N-glycome, O-glycome, glycolipidome, glycoproteome, and proteome providing the most extensive characterization of the glycocalyx between 3D and 2D thus far. The different glycoconjugates were affected in different ways. In the N-glycome, the 3D cells increased in high-mannose glycosylation and in core fucosylation. Glycolipids increased in sialylation. Specific glycoproteins were found to increase in the 3D cell, elucidating the pathways that are affected between the two models. The results show large structural and biological changes between the 2 models suggesting that the 2 are indeed very different potentially affecting individual outcomes in the study of diseases.

Plasma polymers (PPs) can easily modify material surfaces to improve their bio-applicability due to match-made surface-free energy and functionality. However, cell adhesion to PPs typically composed of various functional groups has not yet been fully understood. We explain the origin of strong resistance to trypsin treatment previously noted for nonendothelial cells on amine PPs. It is caused mainly by nonspecific adhesion of negatively charged parts of transmembrane proteins to the positively charged amine PP surface, enabled by thin glycocalyx. However, endothelial cells are bound primarily by their thick, negatively charged glycocalyx and sporadically by integrins in kinetic traps, both cleaved by trypsin. Cell scratching by atomic force microscopy tip confirmed the correlation of trypsin resistance to the strength of cell adhesion.

Sepsis is a condition with high morbidity and mortality. Prompt recognition and initiation of treatment is essential. Despite forming an integral part of sepsis management, fluid resuscitation may also lead to volume overload, which in turn is associated with increased mortality. The optimal fluid strategy in sepsis resuscitation is yet to be defined. Hyaluronan, an endogenous glycosaminoglycan with high affinity to water is an important constituent of the endothelial glycocalyx. We hypothesized that exogenously administered hyaluronan would counteract intravascular volume depletion and contribute to endothelial glycocalyx integrity in a fluid restrictive model of peritonitis. In a prospective, blinded model of porcine peritonitis sepsis, we randomized animals to intervention with hyaluronan (n = 8) or 0.9% saline (n = 8). The animals received an infusion of 0.1% hyaluronan 6 ml/kg/h, or the same volume of saline, during the first 2 h of peritonitis. Stroke volume variation and hemoconcentration were comparable in the two groups throughout the experiment. Cardiac output was higher in the intervention group during the infusion of hyaluronan (3.2 ± 0.5 l/min in intervention group vs 2.7 ± 0.2 l/min in the control group) (p = 0.039). The increase in lactate was more pronounced in the intervention group (3.2 ± 1.0 mmol/l in the intervention group and 1.7 ± 0.7 mmol/l in the control group) at the end of the experiment (p < 0.001). Concentrations of surrogate markers of glycocalyx damage; syndecan 1 (0.6 ± 0.2 ng/ml vs 0.5 ± 0.2 ng/ml, p = 0.292), heparan sulphate (1.23 ± 0.2 vs 1.4 ± 0.3 ng/ml, p = 0.211) and vascular adhesion protein 1 (7.0 ± 4.1 vs 8.2 ± 2.3 ng/ml, p = 0.492) were comparable in the two groups at the end of the experiment. In conclusion, hyaluronan did not counteract intravascular volume depletion in early peritonitis sepsis. However, this finding is hampered by the short observation period and a beneficial effect of HMW-HA in peritonitis sepsis cann, Title in the list of papers of Annelie Barrueta Tenhunen's thesis: Fluid restrictive resuscitation with high molecular weight hyaluronan infusion in early peritonitis-sepsis

Rotavirus (RV) is the leading cause of acute gastroenteritis (AGE) in children under 5 years old worldwide, and several studies have demonstrated that histo-blood group antigens (HBGAs) play a role in its infection process. In the present study, human stool filtrates from patients diagnosed with RV diarrhea (genotyped as P[8]) were used to infect differentiated Caco-2 cells (dCaco-2) to determine whether such viral strains of clinical origin had the ability to replicate in cell cultures displaying HBGAs. The cell culture-adapted human RV Wa model strain (P[8] genotype) was used as a control. A time-course analysis of infection was conducted in dCaco-2 at 1, 24, 48, 72, and 96 h. The replication of two selected clinical isolates and Wa was further assayed in MA104, undifferentiated Caco-2 (uCaco-2), HT29, and HT29-M6 cells, as well as in monolayers of differentiated human intestinal enteroids (HIEs). The results showed that the culture-adapted Wa strain replicated more efficiently in MA104 cells than other utilized cell types. In contrast, clinical virus isolates replicated more efficiently in dCaco-2 cells and HIEs. Furthermore, through surface plasmon resonance analysis of the interaction between the RV spike protein (VP8*) and its glycan receptor (the H antigen), the V7 RV clinical isolate showed 45 times better affinity compared to VP8* from the Wa strain. These findings support the hypothesis that the differences in virus tropism between clinical virus isolates and RV Wa could be a consequence of the different HBGA contents on the surface of the cell lines employed. dCaco-2, HT29, and HT29M6 cells and HIEs display HBGAs on their surfaces, whereas MA104 and uCaco-2 cells do not. These results indicate the relevance of using non-cell culture-adapted human RV to investigate the replication of rotavirus in relevant infection models.

The ability to systematically manipulate the glycocalyx, composed of the collection of glycoconjugates extending from the cell surface, is paramount to understanding structure-function relationships in glycan-mediated biological processes including cell signaling, development, and pathogenesis. Diverse approaches for glycan engineering have emerged, drawing upon chemical, chemoenzymatic, and genetic techniques to augment, edit, or remove cell surface glycoconjugates. Among these strategies, methods to build complexity at the cellular boundary de novo have been particularly effective in revealing the complex relationships between glycan organization within the glycocalyx and the exchange of information between a cell and its environment. While major strides have been made in glycocalyx engineering through the combination of genetic knockouts paired with synthetic glycomaterials that can be inserted into the glycocalyx, these methods generally lack specificity and dynamic control, limiting their biological applications. In this work, several cell surface engineering strategies are reported which collectively address these challenges. First, the ability to spatially program glycan binding activity at the cell surface by scaffolding an artificial glycocalyx comprising both distally presented glycopolymers and surface-proximal glycolipids is presented. Next, utilizing materials bearing photolabile membrane anchors, the ability to spatiotemporally pattern glycocalyx interactions is demonstrated in the context of the mucosal barrier. This glycocalyx photoengineering technique enabled study of the effects of lectin crosslinking on mucin shedding, a key process in pathogenesis. Next, glycomaterials composed of a targeting aptamer unit and bioactive glycodomain were used to program activity in a cell and developmental-stage specific manner in the context of stem cell differentiation, where enhanced differentiation outcomes were observed. Together, these precision glycocalyx e

The diffusion of viruses at the cell membrane is essential to reach a suitable entry site and initiate subsequent internalization. Although many viruses take advantage of glycosaminoglycans (GAG) to bind to the cell surface, little is known about the dynamics of the virus–GAG interactions. Here, single-particle tracking of the initial interaction of individual herpes simplex virus 1 (HSV-1) virions reveals a heterogeneous diffusive behavior, regulated by cell-surface GAGs with two main diffusion types: confined and normal free. This study reports that different GAGs can have competing influences in mediating diffusion on the cells used here: chondroitin sulfate (CS) enhances free diffusion but hinders virus attachment to cell surfaces, while heparan sulfate (HS) promotes virus confinement and increases entry efficiency. In addition, the role that the viral mucin-like domains (MLD) of the HSV-1 glycoprotein C plays in facilitating the diffusion of the virus and accelerating virus penetration into cells is demonstrated. Together, our results shed new light on the mechanisms of GAG-regulated virus diffusion at the cell surface for optimal internalization. These findings may be extendable to other GAG-binding viruses.

The diffusion of viruses at the cell membrane is essential to reach a suitable entry site and initiate subsequent internalization. Although many viruses take advantage of glycosaminoglycans (GAG) to bind to the cell surface, little is known about the dynamics of the virus–GAG interactions. Here, single-particle tracking of the initial interaction of individual herpes simplex virus 1 (HSV-1) virions reveals a heterogeneous diffusive behavior, regulated by cell-surface GAGs with two main diffusion types: confined and normal free. This study reports that different GAGs can have competing influences in mediating diffusion on the cells used here: chondroitin sulfate (CS) enhances free diffusion but hinders virus attachment to cell surfaces, while heparan sulfate (HS) promotes virus confinement and increases entry efficiency. In addition, the role that the viral mucin-like domains (MLD) of the HSV-1 glycoprotein C plays in facilitating the diffusion of the virus and accelerating virus penetration into cells is demonstrated. Together, our results shed new light on the mechanisms of GAG-regulated virus diffusion at the cell surface for optimal internalization. These findings may be extendable to other GAG-binding viruses.

Muscle stem cells (MuSCs) are involved in muscle maintenance and regeneration. Mechanically loaded MuSCs within their native niche undergo tensile and shear deformations, but how MuSCs sense mechanical stimuli and translate these into biochemical signals regulating function and fate is still poorly understood. We aimed to investigate whether the glycocalyx is involved in the MuSC mechanoresponse, and whether MuSC morphology affects mechanical loading-induced pressure, shear stress, and fluid velocity distribution. FSS-induced deformation of active proliferating MuSCs (myoblasts) with intact or degraded glycocalyx was assessed by live-cell imaging. Glycocalyx-degradation did not significantly affect nitric oxide production, but reduced FSS-induced myoblast deformation and modulated gene expression. Finite-element analysis revealed that the distribution of FSS-induced pressure, shear stress, and fluid velocity on myoblasts was non-uniform, and the magnitude depended on myoblast morphology and apex-height. In conclusion, our results suggest that the glycocalyx does not play a role in NO production in myoblasts but might impact mechanotransduction and gene expression, which needs further investigation. Future studies will unravel the underlying mechanism by which the glycocalyx affects FSS-induced myoblast deformation, which might be related to increased drag forces. Moreover, MuSCs with varying apex-height experience different levels of FSS-induced pressure, shear stress, and fluid velocity, suggesting differential responsiveness to fluid shear forces.

The ability to systematically manipulate the glycocalyx, composed of the collection of glycoconjugates extending from the cell surface, is paramount to understanding structure-function relationships in glycan-mediated biological processes including cell signaling, development, and pathogenesis. Diverse approaches for glycan engineering have emerged, drawing upon chemical, chemoenzymatic, and genetic techniques to augment, edit, or remove cell surface glycoconjugates. Among these strategies, methods to build complexity at the cellular boundary de novo have been particularly effective in revealing the complex relationships between glycan organization within the glycocalyx and the exchange of information between a cell and its environment. While major strides have been made in glycocalyx engineering through the combination of genetic knockouts paired with synthetic glycomaterials that can be inserted into the glycocalyx, these methods generally lack specificity and dynamic control, limiting their biological applications. In this work, several cell surface engineering strategies are reported which collectively address these challenges. First, the ability to spatially program glycan binding activity at the cell surface by scaffolding an artificial glycocalyx comprising both distally presented glycopolymers and surface-proximal glycolipids is presented. Next, utilizing materials bearing photolabile membrane anchors, the ability to spatiotemporally pattern glycocalyx interactions is demonstrated in the context of the mucosal barrier. This glycocalyx photoengineering technique enabled study of the effects of lectin crosslinking on mucin shedding, a key process in pathogenesis. Next, glycomaterials composed of a targeting aptamer unit and bioactive glycodomain were used to program activity in a cell and developmental-stage specific manner in the context of stem cell differentiation, where enhanced differentiation outcomes were observed. Together, these precision glycocalyx e

AimsOne of the hallmarks of myocardial infarction (MI) is excessive inflammation. During an inflammatory insult, damaged endothelial cells shed their glycocalyx, a carbohydrate-rich layer on the cell surface which provides a regulatory interface to immune cell adhesion. Selectin-mediated neutrophilia occurs as a result of endothelial injury and inflammation. We recently designed a novel selectin-targeting glycocalyx mimetic (termed DS-IkL) capable of binding inflamed endothelial cells. This study examines the capacity of DS-IkL to limit neutrophil binding and platelet activation on inflamed endothelial cells, as well as the cardioprotective effects of DS-IkL after acute myocardial infarction.Methods and resultsIn vitro, DS-IkL diminished neutrophil interactions with both recombinant selectin and inflamed endothelial cells, and limited platelet activation on inflamed endothelial cells. Our data demonstrated that DS-IkL localized to regions of vascular inflammation in vivo after 45 min of left anterior descending coronary artery ligation-induced MI. Further, findings from this study show DS-IkL treatment had short- and long-term cardioprotective effects after ischaemia/reperfusion of the left anterior descending coronary artery. Mice treated with DS-IkL immediately after ischaemia/reperfusion and 24 h later exhibited reduced neutrophil extravasation, macrophage accumulation, fibroblast and endothelial cell proliferation, and fibrosis compared to saline controls.ConclusionsOur findings suggest that DS-IkL has great therapeutic potential after MI by limiting reperfusion injury induced by the immune response.

Background and Objectives: Preeclampsia is a multisystem disorder that affects maternal endothelium. The glycocalyx lines and protects the endothelial surface. In severe systemic diseases, like sepsis, it is shed and glycocalyx degradation products can be detected in increased concentrations in plasma. The aim of this study was to compare circulating concentrations of glycocalyx degradation products in degrees of preeclampsia severity.Study design: In this observational study, we included women from the South African PROVE biobank. Women were divided into normotensive controls, women with preeclampsia without end-organ complications, women with a single end-organ complication and women with multiple end-organ complications. Plasma samples taken at inclusion after diagnosis (preeclampsia cases) or at admission for delivery (normotensive controls) were analyzed with ELISA for syndecan-1, hyaluronic acid and thrombomodulin and compared between groups.Results: Women with preeclampsia (n = 47) had increased plasma concentrations of hyaluronic acid (100.3 ng/ml IQR 54.2-204 vs. 27.0 ng/ml IQR (13.5-66.6), p < 0,001) and thrombomodulin (4.22 ng/ml IQR 3.55-5.17 vs. 3.49 ng/ml IQR 3.01-3.68, p = 0.007) but not syndecan-1 compared with normotensive women (n = 10). There were no differences in plasma concentration in any of these biomarkers in women with preeclampsia with no end-organ complications (n = 10) compared with women with preeclampsia and one end-organ complication (n = 24). Women with preeclampsia with two or more end-organ complications (n = 13) had increased plasma concentrations of thrombomodulin (5.46 ng/ml, IQR 4.85-7.83 vs. 4.66 ng/ml, IQR 3.45-4.88, p = 0.042) compared with women with preeclampsia and no end-organ complications.Conclusion: Thrombomodulin was associated with disease severity and may be valuable for risk-stratifying women with preeclampsia.

Background and Objectives: Preeclampsia is a multisystem disorder that affects maternal endothelium. The glycocalyx lines and protects the endothelial surface. In severe systemic diseases, like sepsis, it is shed and glycocalyx degradation products can be detected in increased concentrations in plasma. The aim of this study was to compare circulating concentrations of glycocalyx degradation products in degrees of preeclampsia severity.Study design: In this observational study, we included women from the South African PROVE biobank. Women were divided into normotensive controls, women with preeclampsia without end-organ complications, women with a single end-organ complication and women with multiple end-organ complications. Plasma samples taken at inclusion after diagnosis (preeclampsia cases) or at admission for delivery (normotensive controls) were analyzed with ELISA for syndecan-1, hyaluronic acid and thrombomodulin and compared between groups.Results: Women with preeclampsia (n = 47) had increased plasma concentrations of hyaluronic acid (100.3 ng/ml IQR 54.2-204 vs. 27.0 ng/ml IQR (13.5-66.6), p < 0,001) and thrombomodulin (4.22 ng/ml IQR 3.55-5.17 vs. 3.49 ng/ml IQR 3.01-3.68, p = 0.007) but not syndecan-1 compared with normotensive women (n = 10). There were no differences in plasma concentration in any of these biomarkers in women with preeclampsia with no end-organ complications (n = 10) compared with women with preeclampsia and one end-organ complication (n = 24). Women with preeclampsia with two or more end-organ complications (n = 13) had increased plasma concentrations of thrombomodulin (5.46 ng/ml, IQR 4.85-7.83 vs. 4.66 ng/ml, IQR 3.45-4.88, p = 0.042) compared with women with preeclampsia and no end-organ complications.Conclusion: Thrombomodulin was associated with disease severity and may be valuable for risk-stratifying women with preeclampsia.

type:Regular Article, source:https://igakkai.kms-igakkai.com/wp/wp-content/uploads/2021en/KMJ-E202147131.pdf

Despite emerging importance of tumor cells-derived exosomes in cancer metastasis, the heterogeneity of exosome populations has largely hampered systemic characterization of their molecular composition, biogenesis, and functions. This study communicates a novel method for predicting and targeting pre-metastatic sites based on an exosome model fluorescent cancer glyconanosomes displaying N-glycans of cultured tumor cells. Glycoblotting by antiadhesive quantum dots provides a nice tool to shed light on the pivotal functions of the glycocalyx reconstructed from four cancer cell types without bias due to other compositions of exosomes. In vivo imaging revealed that circulation, clearance, and organotropic biodistribution of cancer glyconanosomes in mice depend strongly on cancer cell-type-specific N-glycosylation patterns, the compositions of key glycotypes, particularly dominant abundances of high mannose-type N-glycans and the position-specific sialylation. Notably, organ biodistribution of cancer glyconanosomes is reproducible artificially by mimicking cancer cell-typespecific N-glycosylation patterns, demonstrating that nanosomal glycoblotting method serves as promising tools for predicting and targeting pre-metastatic sites determined by the glycocalyx of extracellular vesicles disseminated from the primary cancer site.

Background and Objectives: Preeclampsia is a multisystem disorder that affects maternal endothelium. The glycocalyx lines and protects the endothelial surface. In severe systemic diseases, like sepsis, it is shed and glycocalyx degradation products can be detected in increased concentrations in plasma. The aim of this study was to compare circulating concentrations of glycocalyx degradation products in degrees of preeclampsia severity.Study design: In this observational study, we included women from the South African PROVE biobank. Women were divided into normotensive controls, women with preeclampsia without end-organ complications, women with a single end-organ complication and women with multiple end-organ complications. Plasma samples taken at inclusion after diagnosis (preeclampsia cases) or at admission for delivery (normotensive controls) were analyzed with ELISA for syndecan-1, hyaluronic acid and thrombomodulin and compared between groups.Results: Women with preeclampsia (n = 47) had increased plasma concentrations of hyaluronic acid (100.3 ng/ml IQR 54.2-204 vs. 27.0 ng/ml IQR (13.5-66.6), p < 0,001) and thrombomodulin (4.22 ng/ml IQR 3.55-5.17 vs. 3.49 ng/ml IQR 3.01-3.68, p = 0.007) but not syndecan-1 compared with normotensive women (n = 10). There were no differences in plasma concentration in any of these biomarkers in women with preeclampsia with no end-organ complications (n = 10) compared with women with preeclampsia and one end-organ complication (n = 24). Women with preeclampsia with two or more end-organ complications (n = 13) had increased plasma concentrations of thrombomodulin (5.46 ng/ml, IQR 4.85-7.83 vs. 4.66 ng/ml, IQR 3.45-4.88, p = 0.042) compared with women with preeclampsia and no end-organ complications.Conclusion: Thrombomodulin was associated with disease severity and may be valuable for risk-stratifying women with preeclampsia.

Background and Objectives: Preeclampsia is a multisystem disorder that affects maternal endothelium. The glycocalyx lines and protects the endothelial surface. In severe systemic diseases, like sepsis, it is shed and glycocalyx degradation products can be detected in increased concentrations in plasma. The aim of this study was to compare circulating concentrations of glycocalyx degradation products in degrees of preeclampsia severity.Study design: In this observational study, we included women from the South African PROVE biobank. Women were divided into normotensive controls, women with preeclampsia without end-organ complications, women with a single end-organ complication and women with multiple end-organ complications. Plasma samples taken at inclusion after diagnosis (preeclampsia cases) or at admission for delivery (normotensive controls) were analyzed with ELISA for syndecan-1, hyaluronic acid and thrombomodulin and compared between groups.Results: Women with preeclampsia (n = 47) had increased plasma concentrations of hyaluronic acid (100.3 ng/ml IQR 54.2-204 vs. 27.0 ng/ml IQR (13.5-66.6), p < 0,001) and thrombomodulin (4.22 ng/ml IQR 3.55-5.17 vs. 3.49 ng/ml IQR 3.01-3.68, p = 0.007) but not syndecan-1 compared with normotensive women (n = 10). There were no differences in plasma concentration in any of these biomarkers in women with preeclampsia with no end-organ complications (n = 10) compared with women with preeclampsia and one end-organ complication (n = 24). Women with preeclampsia with two or more end-organ complications (n = 13) had increased plasma concentrations of thrombomodulin (5.46 ng/ml, IQR 4.85-7.83 vs. 4.66 ng/ml, IQR 3.45-4.88, p = 0.042) compared with women with preeclampsia and no end-organ complications.Conclusion: Thrombomodulin was associated with disease severity and may be valuable for risk-stratifying women with preeclampsia.

The diffusion of viruses at the cell membrane is essential to reach a suitable entry site and initiate subsequent internalization. Although many viruses take advantage of glycosaminoglycans (GAG) to bind to the cell surface, little is known about the dynamics of the virus–GAG interactions. Here, single-particle tracking of the initial interaction of individual herpes simplex virus 1 (HSV-1) virions reveals a heterogeneous diffusive behavior, regulated by cell-surface GAGs with two main diffusion types: confined and normal free. This study reports that different GAGs can have competing influences in mediating diffusion on the cells used here: chondroitin sulfate (CS) enhances free diffusion but hinders virus attachment to cell surfaces, while heparan sulfate (HS) promotes virus confinement and increases entry efficiency. In addition, the role that the viral mucin-like domains (MLD) of the HSV-1 glycoprotein C plays in facilitating the diffusion of the virus and accelerating virus penetration into cells is demonstrated. Together, our results shed new light on the mechanisms of GAG-regulated virus diffusion at the cell surface for optimal internalization. These findings may be extendable to other GAG-binding viruses.

Vascular dysfunction and organ failure are two distinct, albeit highly interconnected, clinical outcomes linked to morbidity and mortality in human sepsis. The mechanisms driving vascular and parenchymal damage are dynamic and display significant molecular cross talk between organs and tissues. Therefore, assessing their individual contribution to disease progression is technically challenging. Here, we hypothesize that dysregulated vascular responses predispose the organism to organ failure. To address this hypothesis, we have evaluated four major organs in a murine model of Staphylococcus aureus sepsis by combining in vivo labeling of the endothelial cell surface proteome, data-independent acquisition (DIA) mass spectrometry, and an integrative computational pipeline. The data reveal, with unprecedented depth and throughput, that a septic insult evokes organ-specific proteome responses that are highly compartmentalized, synchronously coordinated, and significantly correlated with the progression of the disease. These responses include abundant vascular shedding, dysregulation of the intrinsic pathway of coagulation, compartmentalization of the acute phase response, and abundant upregulation of glycocalyx components. Vascular cell surface proteome changes were also found to precede bacterial invasion and leukocyte infiltration into the organs, as well as to precede changes in various well-established cellular and biochemical correlates of systemic coagulopathy and tissue dysfunction. Importantly, our data suggest a potential role for the vascular proteome as a determinant of the susceptibility of the organs to undergo failure during sepsis. IMPORTANCE Sepsis is a life-threatening response to infection that results in immune dysregulation, vascular dysfunction, and organ failure. New methods are needed for the identification of diagnostic and therapeutic targets. Here, we took a systems-wide approach using data-independent acquisition (DIA) mass spectrometry to trac

Despite emerging importance of tumor cells-derived exosomes in cancer metastasis, the heterogeneity of exosome populations has largely hampered systemic characterization of their molecular composition, biogenesis, and functions. This study communicates a novel method for predicting and targeting pre-metastatic sites based on an exosome model fluorescent cancer glyconanosomes displaying N-glycans of cultured tumor cells. Glycoblotting by antiadhesive quantum dots provides a nice tool to shed light on the pivotal functions of the glycocalyx reconstructed from four cancer cell types without bias due to other compositions of exosomes. In vivo imaging revealed that circulation, clearance, and organotropic biodistribution of cancer glyconanosomes in mice depend strongly on cancer cell-type-specific N-glycosylation patterns, the compositions of key glycotypes, particularly dominant abundances of high mannose-type N-glycans and the position-specific sialylation. Notably, organ biodistribution of cancer glyconanosomes is reproducible artificially by mimicking cancer cell-typespecific N-glycosylation patterns, demonstrating that nanosomal glycoblotting method serves as promising tools for predicting and targeting pre-metastatic sites determined by the glycocalyx of extracellular vesicles disseminated from the primary cancer site.

Marginal liver grafts, such as steatotic livers and those from cardiac death donors, are highly vulnerable to ischemia–reperfusion injury that occurs in the complex route of the graft from “harvest to revascularization”. Recently, several preservation methods have been developed to preserve liver grafts based on hypothermic static preservation and hypothermic oxygenated perfusion (HOPE) strategies, either combined or alone. However, their effects on mitochondrial functions and their relevance have not yet been fully investigated, especially if different preservation solutions/effluents are used. Ischemic liver graft damage is caused by oxygen deprivation conditions during cold storage that provoke alterations in mitochondrial integrity and function and energy metabolism breakdown. This review deals with the relevance of mitochondrial machinery in cold static preservation and how the mitochondrial respiration function through the accumulation of succinate at the end of cold ischemia is modulated by different preservation solutions such as IGL-2, HTK, and UW (gold-standard reference). IGL-2 increases mitochondrial integrity and function (ALDH2) when compared to UW and HTK. This mitochondrial protection by IGL-2 also extends to protective HOPE strategies when used as an effluent instead of Belzer MP. The transient oxygenation in HOPE sustains the mitochondrial machinery at basal levels and prevents, in part, the accumulation of energy metabolites such as succinate in contrast to those that occur in cold static preservation conditions. Additionally, several additives for combating oxygen deprivation and graft energy metabolism breakdown during hypothermic static preservation such as oxygen carriers, ozone, AMPK inducers, and mitochondrial UCP2 inhibitors, and whether they are or not to be combined with HOPE, are presented and discussed. Finally, we affirm that IGL-2 solution is suitable for protecting graft mitochondrial machinery and simplifying the complex logistics i

The scarcity of livers for transplantation is rising, and new strategies to extend the donor pool are being explored. One solution is to use marginal grafts from extended criteria donors, presenting, for example, liver steatosis. As current preservation solutions (UW, HTK, and IGL-1) were mainly designed for static cold storage (SCS) only, IGL-2, a modified version of IGL-1, was developed to be suitable for SCS and dynamic preservation, such as hypothermic oxygenated perfusion (HOPE). In this study, we investigated the combined effect of IGL-2, SCS, and HOPE and compared it to the most used preservation solution (UW and Belzer MPS). Four experimental groups with six rats each were designed using Zucker rats. All groups underwent 24 h of SCS (in IGL-2 or UW) + 2 h of normothermic machine perfusion (NMP) at 37 °C to mimic transplantation. HOPE (IGL-2 or Belzer MPS) was performed before NMP on half of the rats. The IGL-2 group demonstrated lower transaminases and a significantly low level of glycocalyx proteins, CASP3, and HMGB1 in the perfusates. These data suggest the protective role of IGL-2 for fatty livers in preserving the endothelial glycocalyx, apoptosis, and inflammation.

Vascular dysfunction and organ failure are two distinct, albeit highly interconnected, clinical outcomes linked to morbidity and mortality in human sepsis. The mechanisms driving vascular and parenchymal damage are dynamic and display significant molecular cross talk between organs and tissues. Therefore, assessing their individual contribution to disease progression is technically challenging. Here, we hypothesize that dysregulated vascular responses predispose the organism to organ failure. To address this hypothesis, we have evaluated four major organs in a murine model of Staphylococcus aureus sepsis by combining in vivo labeling of the endothelial cell surface proteome, data-independent acquisition (DIA) mass spectrometry, and an integrative computational pipeline. The data reveal, with unprecedented depth and throughput, that a septic insult evokes organ-specific proteome responses that are highly compartmentalized, synchronously coordinated, and significantly correlated with the progression of the disease. These responses include abundant vascular shedding, dysregulation of the intrinsic pathway of coagulation, compartmentalization of the acute phase response, and abundant upregulation of glycocalyx components. Vascular cell surface proteome changes were also found to precede bacterial invasion and leukocyte infiltration into the organs, as well as to precede changes in various well-established cellular and biochemical correlates of systemic coagulopathy and tissue dysfunction. Importantly, our data suggest a potential role for the vascular proteome as a determinant of the susceptibility of the organs to undergo failure during sepsis.

The diffusion of viruses at the cell membrane is essential to reach a suitable entry site and initiate subsequent internalization. Although many viruses take advantage of glycosaminoglycans (GAG) to bind to the cell surface, little is known about the dynamics of the virus–GAG interactions. Here, single-particle tracking of the initial interaction of individual herpes simplex virus 1 (HSV-1) virions reveals a heterogeneous diffusive behavior, regulated by cell-surface GAGs with two main diffusion types: confined and normal free. This study reports that different GAGs can have competing influences in mediating diffusion on the cells used here: chondroitin sulfate (CS) enhances free diffusion but hinders virus attachment to cell surfaces, while heparan sulfate (HS) promotes virus confinement and increases entry efficiency. In addition, the role that the viral mucin-like domains (MLD) of the HSV-1 glycoprotein C plays in facilitating the diffusion of the virus and accelerating virus penetration into cells is demonstrated. Together, our results shed new light on the mechanisms of GAG-regulated virus diffusion at the cell surface for optimal internalization. These findings may be extendable to other GAG-binding viruses.

The cell/microenvironment interface is the starting point of integrin-mediated mechanotransduction, but many details of mechanotransductive signal integration remain elusive due to the complexity of the involved (extra)cellular structures, such as the glycocalyx. We used nano-bio-interfaces reproducing the complex nanotopographical features of the extracellular matrix to analyse the glycocalyx impact on PC12 cell mechanosensing at the nanoscale (e.g., by force spectroscopy with functionalised probes). Our data demonstrates that the glycocalyx configuration affects spatio-temporal nanotopography-sensitive mechanotransductive events at the cell/microenvironment interface. Opposing effects of major glycocalyx removal were observed, when comparing flat and specific nanotopographical conditions. The excessive retrograde actin flow speed and force loading are strongly reduced on certain nanotopographies upon strong reduction of the native glycocalyx, while on the flat substrate we observe the opposite trend. Our results highlight the importance of the glycocalyx configuration in a molecular clutch force loading-dependent cellular mechanism for mechanosensing of microenvironmental nanotopographical features.

Background and Objectives: Preeclampsia is a multisystem disorder that affects maternal endothelium. The glycocalyx lines and protects the endothelial surface. In severe systemic diseases, like sepsis, it is shed and glycocalyx degradation products can be detected in increased concentrations in plasma. The aim of this study was to compare circulating concentrations of glycocalyx degradation products in degrees of preeclampsia severity.Study design: In this observational study, we included women from the South African PROVE biobank. Women were divided into normotensive controls, women with preeclampsia without end-organ complications, women with a single end-organ complication and women with multiple end-organ complications. Plasma samples taken at inclusion after diagnosis (preeclampsia cases) or at admission for delivery (normotensive controls) were analyzed with ELISA for syndecan-1, hyaluronic acid and thrombomodulin and compared between groups.Results: Women with preeclampsia (n = 47) had increased plasma concentrations of hyaluronic acid (100.3 ng/ml IQR 54.2-204 vs. 27.0 ng/ml IQR (13.5-66.6), p < 0,001) and thrombomodulin (4.22 ng/ml IQR 3.55-5.17 vs. 3.49 ng/ml IQR 3.01-3.68, p = 0.007) but not syndecan-1 compared with normotensive women (n = 10). There were no differences in plasma concentration in any of these biomarkers in women with preeclampsia with no end-organ complications (n = 10) compared with women with preeclampsia and one end-organ complication (n = 24). Women with preeclampsia with two or more end-organ complications (n = 13) had increased plasma concentrations of thrombomodulin (5.46 ng/ml, IQR 4.85-7.83 vs. 4.66 ng/ml, IQR 3.45-4.88, p = 0.042) compared with women with preeclampsia and no end-organ complications.Conclusion: Thrombomodulin was associated with disease severity and may be valuable for risk-stratifying women with preeclampsia.

The diffusion of viruses at the cell membrane is essential to reach a suitable entry site and initiate subsequent internalization. Although many viruses take advantage of glycosaminoglycans (GAG) to bind to the cell surface, little is known about the dynamics of the virus–GAG interactions. Here, single-particle tracking of the initial interaction of individual herpes simplex virus 1 (HSV-1) virions reveals a heterogeneous diffusive behavior, regulated by cell-surface GAGs with two main diffusion types: confined and normal free. This study reports that different GAGs can have competing influences in mediating diffusion on the cells used here: chondroitin sulfate (CS) enhances free diffusion but hinders virus attachment to cell surfaces, while heparan sulfate (HS) promotes virus confinement and increases entry efficiency. In addition, the role that the viral mucin-like domains (MLD) of the HSV-1 glycoprotein C plays in facilitating the diffusion of the virus and accelerating virus penetration into cells is demonstrated. Together, our results shed new light on the mechanisms of GAG-regulated virus diffusion at the cell surface for optimal internalization. These findings may be extendable to other GAG-binding viruses.

Background: In a pilot study, we found a significant reduction in mean daily sequential organ failure assessment score in mechanically ventilated patients with COVID-19 who received prostacyclin, compared to placebo. We here investigate the effect on biomarkers of endothelial activation and damage. Methods: Post-hoc study of a randomized controlled trial in adult patients with confirmed SARS-CoV-2 infection, mechanically ventilated, with soluble thrombomodulin (sTM) plasma levels >4 ng/mL. Patients received prostacyclin infusion (1 ng/kg/min) or placebo. Blood samples were collected at baseline and 24 h. Results: Eighty patients were randomized (41 prostacyclin, 39 placebo). The median changes in syndecan-1 plasma levels at 24 h were −3.95 (IQR: −21.1 to 2.71) ng/mL in the prostacyclin group vs. 3.06 (IQR: −8.73 to 20.5) ng/mL in the placebo group (difference of the medians: -7.01 [95% CI: −22.3 to −0.231] ng/mL, corresponding to −3% [95% CI: −11% to 0%], p = 0.04). Changes in plasma levels of sTM, PECAM-1, p-selectin, and CD40L did not differ significantly between groups. Conclusions: Prostacyclin infusion, compared to placebo, resulted in a measurable decrease in endothelial glycocalyx shedding (syndecan-1) at 24 h, suggesting a protective effect on the endothelium, which may be related to the observed reduction in organ failure.

N-glycosylation is a ubiquitous posttranslational modification that affects protein structure and function, including those of the central nervous system. N-glycans attached to cell membrane proteins play crucial roles in all aspects of biology, including embryogenesis, development, cell-cell recognition and adhesion, and cell signaling and communication. Although brain function and behavior are known to be regulated by the N-glycosylation state of numerous cell surface glycoproteins, our current understanding of brain glycosylation is limited, and glycan variations associated with functional brain regions remain largely unknown. In this work, we used a well-established cell surface glycomic nanoLC-Chip-Q-TOF platform developed in our laboratory to characterize the N-glycome of membrane fractions enriched in cell surface glycoproteins obtained from specific functional brain areas. We report the cell membrane N-glycome of two major developmental divisions of mice brain with specific and distinctive functions, namely the forebrain and hindbrain. Region-specific glycan maps were obtained with ∼120 N-glycan compositions in each region, revealing significant differences in "brain-type" glycans involving high mannose, bisecting, and core and antenna fucosylated species. Additionally, the cell membrane N-glycome of three functional regions of the forebrain and hindbrain, the cerebral cortex, hippocampus, and cerebellum, was characterized. In total, 125 N-glycan compositions were identified, and their region-specific expression profiles were characterized. Over 70 N-glycans contributed to the differentiation of the cerebral cortex, hippocampus, and cerebellum N-glycome, including bisecting and branched glycans with varying degrees of core and antenna fucosylation and sialylation. This study presents a comprehensive spatial distribution of the cell-membrane enriched N-glycomes associated with five discrete anatomical and functional brain areas, providing evidence for the pre

N-glycosylation is a ubiquitous posttranslational modification that affects protein structure and function, including those of the central nervous system. N-glycans attached to cell membrane proteins play crucial roles in all aspects of biology, including embryogenesis, development, cell-cell recognition and adhesion, and cell signaling and communication. Although brain function and behavior are known to be regulated by the N-glycosylation state of numerous cell surface glycoproteins, our current understanding of brain glycosylation is limited, and glycan variations associated with functional brain regions remain largely unknown. In this work, we used a well-established cell surface glycomic nanoLC-Chip-Q-TOF platform developed in our laboratory to characterize the N-glycome of membrane fractions enriched in cell surface glycoproteins obtained from specific functional brain areas. We report the cell membrane N-glycome of two major developmental divisions of mice brain with specific and distinctive functions, namely the forebrain and hindbrain. Region-specific glycan maps were obtained with ∼120 N-glycan compositions in each region, revealing significant differences in "brain-type" glycans involving high mannose, bisecting, and core and antenna fucosylated species. Additionally, the cell membrane N-glycome of three functional regions of the forebrain and hindbrain, the cerebral cortex, hippocampus, and cerebellum, was characterized. In total, 125 N-glycan compositions were identified, and their region-specific expression profiles were characterized. Over 70 N-glycans contributed to the differentiation of the cerebral cortex, hippocampus, and cerebellum N-glycome, including bisecting and branched glycans with varying degrees of core and antenna fucosylation and sialylation. This study presents a comprehensive spatial distribution of the cell-membrane enriched N-glycomes associated with five discrete anatomical and functional brain areas, providing evidence for the pre

After an acute inflammatory event such as a heart attack, stroke, or organ transplant, blood vessels and circulating immune cells take on a complex phenotype that, if dysregulated, can perpetuate disease. Adverse changes in endothelial phenotype, including glycocalyx shedding and upregulation of immune cell adhesion molecules (i.e., selectins), facilitate the recruitment and adhesion of circulating leukocytes (i.e., neutrophils) and platelets to the vessel walls. In some cases, this otherwise restorative physiological response devolves into a proinflammatory feedback loop; this process is shared among organ systems, enabling disease-agnostic drug discovery. Selectins are an attractive therapeutic target to inhibit the proinflammatory feedback loop; however, their low affinity for monovalent binding ligands and pivotal role in restorative inflammatory mechanisms has created a barrier to drug development. In addition, glycosaminoglycans such as dermatan sulfate (DS) have inherent anti-inflammatory properties that, if localized to a pro-inflammatory environment, can indirectly regulate immune cell adhesion by creating a steric, band-aid-like surface along inflamed endothelial cells. Through the chapters of this dissertation, we introduce a novel selectin-binding glycosaminoglycan-peptide conjugate (DS-IkL) as a molecular “band-aid” to address multiple elements of endothelial dysfunction, including: selectin upregulation, glycocalyx shedding, and aberrant immune cell adhesion and influx. We show that DS-IkL localizes to inflamed vasculature, interferes with selectin-mediated cell complexing, reduces neutrophil extravasation into tissue, and has short- and long-term cardioprotective effects following an acute myocardial infarction. This work details the (i) discovery of a novel selectin-targeting peptide comprised of both L- and D-amino acids and its tunable conjugation to a DS polymer; (ii) functional characterization of DS-IkL’s capacity to curb neutrophil binding, pla

Glycosylation is one of the most prevalent post-translational modifications found on biomolecules that regulate various biological functions such as cell-cell interactions, immune responses, cellular regulations, viral/bacterial infections, and much more. Aberration in glycans can lead to alterations in protein function and have been shown to occur in many diseases such as cancer. In recent years, a few glycan-based biomarkers for cancer have been discovered. However, cancer is still a leading cause of death worldwide. Therefore, additional effort is needed to understand the cause of cancer and how glycosylation plays a role in cancer prognosis. This dissertation presents a series of state-of-the-art nano-liquid chromatography-mass spectrometry (nanoLC-MS) methods and bioinformatics tools to characterize the glycocalyx. In Chapter I, a thorough introduction of glycan synthesis, glycan biological functions, and methods of glycan analysis are presented. Chapter II presents the alteration of the N-glycome of several cancer cell lines through metabolic engineering using glycosylation inhibitors. Chapter III presents the first extensive “omic” analysis of the glycocalyx of 3D cell culture of cancer to elucidate alteration in the glycocalyx relative to its 2D counterpart. Finally, chapter IV presents the characterization of glycocalyx of lung cancer cell lines treated with a therapeutic drug to monitor glycocalyx alteration.

Background: Plasma hyaluronan concentrations are increased during sepsis but underlying mechanisms leading to high plasma hyaluronan concentration are poorly understood. In this study we evaluate the roles of plasma hyaluronan, effective plasma hyaluronidase (HYAL) activity and its endogenous plasma inhibition in clinical and experimental sepsis. We specifically hypothesized that plasma HYAL acts as endothelial glycocalyx shedding enzyme, sheddase. Methods: Plasma hyaluronan, effective HYAL activity and HYAL inhibition were measured in healthy volunteers (n = 20), in patients with septic shock (n = 17, day 1 and day 4), in patients with acute pancreatitis (n = 7, day 1 and day 4) and in anesthetized and mechanically ventilated pigs (n = 16). Sixteen pigs were allocated (unblinded, open label) into three groups: Sepsis-1 with infusion of live Escherichia coli (E. coli) 1 x 10(8) CFU/h of 12 h (n = 5), Sepsis-2 with infusion of E. coli 1 x 10(8) CFU/h of 6 h followed by 1 x 10(9) CFU/h of the remaining 6 h (n = 5) or Control with no E. coli infusion (n = 6). Results: In experimental E. coli porcine sepsis and in time controls, plasma hyaluronan increases with concomitant decrease in effective plasma HYAL activity and increase of endogenous HYAL inhibition. Plasma hyaluronan increased in patients with septic shock but not in acute pancreatitis. Effective plasma HYAL was lower in septic shock and acute pancreatitis as compared to healthy volunteers, while plasma HYAL inhibition was only increased in septic shock. Conclusion: Elevated plasma hyaluronan levels coincided with a concomitant decrease in effective plasma HYAL activity and increase of endogenous plasma HYAL inhibition both in experimental and clinical sepsis. In acute pancreatitis, effective plasma HYAL activity was decreased which was not associated with increased plasma hyaluronan concentrations or endogenous HYAL inhibition. The results suggest that plasma HYAL does not act as sheddase in sepsis or pancreat

Background: Plasma hyaluronan concentrations are increased during sepsis but underlying mechanisms leading to high plasma hyaluronan concentration are poorly understood. In this study we evaluate the roles of plasma hyaluronan, effective plasma hyaluronidase (HYAL) activity and its endogenous plasma inhibition in clinical and experimental sepsis. We specifically hypothesized that plasma HYAL acts as endothelial glycocalyx shedding enzyme, sheddase. Methods: Plasma hyaluronan, effective HYAL activity and HYAL inhibition were measured in healthy volunteers (n = 20), in patients with septic shock (n = 17, day 1 and day 4), in patients with acute pancreatitis (n = 7, day 1 and day 4) and in anesthetized and mechanically ventilated pigs (n = 16). Sixteen pigs were allocated (unblinded, open label) into three groups: Sepsis-1 with infusion of live Escherichia coli (E. coli) 1 x 10(8) CFU/h of 12 h (n = 5), Sepsis-2 with infusion of E. coli 1 x 10(8) CFU/h of 6 h followed by 1 x 10(9) CFU/h of the remaining 6 h (n = 5) or Control with no E. coli infusion (n = 6). Results: In experimental E. coli porcine sepsis and in time controls, plasma hyaluronan increases with concomitant decrease in effective plasma HYAL activity and increase of endogenous HYAL inhibition. Plasma hyaluronan increased in patients with septic shock but not in acute pancreatitis. Effective plasma HYAL was lower in septic shock and acute pancreatitis as compared to healthy volunteers, while plasma HYAL inhibition was only increased in septic shock. Conclusion: Elevated plasma hyaluronan levels coincided with a concomitant decrease in effective plasma HYAL activity and increase of endogenous plasma HYAL inhibition both in experimental and clinical sepsis. In acute pancreatitis, effective plasma HYAL activity was decreased which was not associated with increased plasma hyaluronan concentrations or endogenous HYAL inhibition. The results suggest that plasma HYAL does not act as sheddase in sepsis or pancreat

Background: Plasma hyaluronan concentrations are increased during sepsis but underlying mechanisms leading to high plasma hyaluronan concentration are poorly understood. In this study we evaluate the roles of plasma hyaluronan, effective plasma hyaluronidase (HYAL) activity and its endogenous plasma inhibition in clinical and experimental sepsis. We specifically hypothesized that plasma HYAL acts as endothelial glycocalyx shedding enzyme, sheddase. Methods: Plasma hyaluronan, effective HYAL activity and HYAL inhibition were measured in healthy volunteers (n = 20), in patients with septic shock (n = 17, day 1 and day 4), in patients with acute pancreatitis (n = 7, day 1 and day 4) and in anesthetized and mechanically ventilated pigs (n = 16). Sixteen pigs were allocated (unblinded, open label) into three groups: Sepsis-1 with infusion of live Escherichia coli (E. coli) 1 x 10(8) CFU/h of 12 h (n = 5), Sepsis-2 with infusion of E. coli 1 x 10(8) CFU/h of 6 h followed by 1 x 10(9) CFU/h of the remaining 6 h (n = 5) or Control with no E. coli infusion (n = 6). Results: In experimental E. coli porcine sepsis and in time controls, plasma hyaluronan increases with concomitant decrease in effective plasma HYAL activity and increase of endogenous HYAL inhibition. Plasma hyaluronan increased in patients with septic shock but not in acute pancreatitis. Effective plasma HYAL was lower in septic shock and acute pancreatitis as compared to healthy volunteers, while plasma HYAL inhibition was only increased in septic shock. Conclusion: Elevated plasma hyaluronan levels coincided with a concomitant decrease in effective plasma HYAL activity and increase of endogenous plasma HYAL inhibition both in experimental and clinical sepsis. In acute pancreatitis, effective plasma HYAL activity was decreased which was not associated with increased plasma hyaluronan concentrations or endogenous HYAL inhibition. The results suggest that plasma HYAL does not act as sheddase in sepsis or pancreat

After an acute inflammatory event such as a heart attack, stroke, or organ transplant, blood vessels and circulating immune cells take on a complex phenotype that, if dysregulated, can perpetuate disease. Adverse changes in endothelial phenotype, including glycocalyx shedding and upregulation of immune cell adhesion molecules (i.e., selectins), facilitate the recruitment and adhesion of circulating leukocytes (i.e., neutrophils) and platelets to the vessel walls. In some cases, this otherwise restorative physiological response devolves into a proinflammatory feedback loop; this process is shared among organ systems, enabling disease-agnostic drug discovery. Selectins are an attractive therapeutic target to inhibit the proinflammatory feedback loop; however, their low affinity for monovalent binding ligands and pivotal role in restorative inflammatory mechanisms has created a barrier to drug development. In addition, glycosaminoglycans such as dermatan sulfate (DS) have inherent anti-inflammatory properties that, if localized to a pro-inflammatory environment, can indirectly regulate immune cell adhesion by creating a steric, band-aid-like surface along inflamed endothelial cells. Through the chapters of this dissertation, we introduce a novel selectin-binding glycosaminoglycan-peptide conjugate (DS-IkL) as a molecular “band-aid” to address multiple elements of endothelial dysfunction, including: selectin upregulation, glycocalyx shedding, and aberrant immune cell adhesion and influx. We show that DS-IkL localizes to inflamed vasculature, interferes with selectin-mediated cell complexing, reduces neutrophil extravasation into tissue, and has short- and long-term cardioprotective effects following an acute myocardial infarction. This work details the (i) discovery of a novel selectin-targeting peptide comprised of both L- and D-amino acids and its tunable conjugation to a DS polymer; (ii) functional characterization of DS-IkL’s capacity to curb neutrophil binding, pla

Glycosylation is one of the most prevalent post-translational modifications found on biomolecules that regulate various biological functions such as cell-cell interactions, immune responses, cellular regulations, viral/bacterial infections, and much more. Aberration in glycans can lead to alterations in protein function and have been shown to occur in many diseases such as cancer. In recent years, a few glycan-based biomarkers for cancer have been discovered. However, cancer is still a leading cause of death worldwide. Therefore, additional effort is needed to understand the cause of cancer and how glycosylation plays a role in cancer prognosis. This dissertation presents a series of state-of-the-art nano-liquid chromatography-mass spectrometry (nanoLC-MS) methods and bioinformatics tools to characterize the glycocalyx. In Chapter I, a thorough introduction of glycan synthesis, glycan biological functions, and methods of glycan analysis are presented. Chapter II presents the alteration of the N-glycome of several cancer cell lines through metabolic engineering using glycosylation inhibitors. Chapter III presents the first extensive “omic” analysis of the glycocalyx of 3D cell culture of cancer to elucidate alteration in the glycocalyx relative to its 2D counterpart. Finally, chapter IV presents the characterization of glycocalyx of lung cancer cell lines treated with a therapeutic drug to monitor glycocalyx alteration.