Glycocalyx Research Articles

Detection of Glycan Shedding in the Blood: New Class of Multiple Sclerosis Biomarkers?

Multiple sclerosis (MS) is a devastating autoimmune disease, afflicting people in the prime of their lives. Presently, after initial clinical presentation, there are no reliable markers for whether a patient will develop MS, or whether their prognosis will be aggressive or relapsing-remitting. Furthermore, many MS patients do not respond to treatment. Thus, markers for diagnosis, prognosis, and treatment-responsiveness are lacking for a disease, where a precision medicine approach would be valuable. The glycocalyx (GLX) is the carbohydrate-rich outer surface of the blood vessel wall and is the first interaction between the blood and the vessel. We hypothesized that cleavage of the GLX may be an early stage predictor of immune attack, blood-brain barrier (BBB) breakdown, and disease severity in MS. Two experimental models of MS, experimental autoimmune encephalitis (EAE), were included in this study. EAE was induced in C57BL/6J mice and Lewis rats, which were monitored for weight loss and clinical presentation in comparison to healthy controls. Plasma samples were obtained longitudinally from mice until peak disease severity and at peak disease severity in rats. Soluble GLX-associated glycosaminoglycans (GAG) and proteoglycans (PG) were detected in plasma samples. All animals receiving EAE emulsion developed fulminant EAE (100% penetrance). Increased plasma levels of chondroitin sulfate were detected before the onset of clinical symptoms and remained elevated at peak disease severity. Hyaluronic acid was increased at the height of the disease, whereas heparan sulfate was transiently increased during early stages only. By contrast, syndecans 1, 3, and 4 were detected in EAE samples as well as healthy controls, with no significant differences between the two groups. In this study, we present data supporting the shedding of the GLX as a new class of biomarker for MS. In particular, soluble, sugar-based GLX components are associated with disease severity in two models of MS, molecules that would not be detected in proteomics-based screens of MS patient samples. Patient studies are presently underway.

Redox regulation in human endothelial cells: a critical role for the glycocalyx in mechanotransduction of fluid shear stress

Redox status in endothelial cells (EC) is focally regulated by fluid shear stress (FSS) forces acting on the vessel lumen. Notably, endogenous antioxidant genes targeted by the redox sensitive transcription factor nuclear factor E2-related factor 2 (Nrf2) are upregulated by unidirectional (US) FSS. The glycocalyx (GCX), a meshwork of glycosaminoglycans, is prominent in atheroprotected EC but scarce in atherosusceptible EC regions, and a prerequisite for FSS induced nitric oxide and reactive oxygen species generation. In this study, we investigate the role of the GCX as a modulator of redox signaling in human EC (HUVEC) subjected to either US (15 dynes/cm2) or oscillatory (±5 dynes/cm2, 1 Hz) shear for 48 h in microfluidic slides (Ibidi GmbH). The sialic acid (SA) component of the GCX and expression of heme oxygenase-1 (HO-1) were differentially regulated by US and OS compared to static cultures. Removal of SA with neuraminidase (2U/ml, 30 min) prior to US exposure abrogated HO-1 induction and enhanced mitochondrial superoxide in response to FSS. These findings demonstrate that FSS-sensitive Nrf2 signaling is dependent on mechanotransduction through the GCX, and restoration of the GCX may normalise redox homeostasis in atheroprone vascular regions.

Coating of Single Cells towards Biomedical Applications

In 2002, a pioneering work on single cell coating with macromolecules through layer-by-layer...

Sequential Coating of Insulin Secreting Beta Cells within Multilayers of Polysaccharide Nanogels

Pancreatic islet transplantation has emerged as a promising treatment for type-1 diabetes (T1D); however, its clinical application is still limited by the life-long use of immunosuppressive drugs, insufficient number of islets to achieve normoglycemia, and large transplantation volume. This paper reports a unique approach for nanothin coating of insulin secreting beta cell aggregates. The coating is based on hydrophobic and covalent interactions between natural acrylate modified cholesterol bearing pullulan (CHPOA) nanogels and MIN6 beta cell aggregates. Beta cell aggregates are prepared as spheroids through hanging drop method, which is optimized with respect to hanging drop volume and initial number of beta cells. These aggregates, defined as pseudoislets, are coated with sequential layers of nanogels and are evaluated as viable and functional for insulin secretion. Coating experiments are carried out using physiologically compatible medium, where pseudoislets are not brought in contact with toxic prepolymer solutions used in existing approaches. This study offers new opportunities through coating of islets with advanced functional materials under completely physiological conditions for clinical translation of cell transplantation technology. The technique developed here will establish a new paradigm for creating tolerable grafts for other chronic diseases such as anemia, cancer, central nervous system (CNS) diseases.

Study on expression of plasma sCD138 in patients with hemorrhagic fever with renal syndrome

BackgroundUntil now, there is non-specific treatment, and exploring early and novel biomarkers to determine the disease severity and prognosis of hemorrhagic fever with renal syndrome (HFRS) would be of importance for clinician to take systematic and timely intervention. This study observed the expression of plasma sCD138, a soluble component shedding from the glycocalyx (GCX) to the circulating blood, and evaluated its predictive value on disease severity and prognosis of HFRS.MethodsOne hundred and seventy-six patients with HFRS who were treated at our center between January 2011 and December 2013 were randomly enrolled in this study. The patients were divided into a mild-type group, a moderate-type group, a severe-type group and a critical-type group according to the HFRS criteria for clinical classification. Thirty-five blood samples from healthy subjects were obtained as the controls. The concentrations of sCD138 were detected using enzyme linked immunosorbent assay (ELISA). The levels of prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (Fib), albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), white blood cells (WBC), platelets (PLT), glucose (GLU), blood urea nitrogen (BUN) and serum creatinine (Scr) in the samples were routinely tested. The levels of sCD138 among the different types were compared; the correlation among sCD138 and the laboratory parameters mentioned above were analyzed. The predictive effectiveness for prognosis of sCD138 was evaluated using the receiver operating characteristic (ROC) curve analysis.ResultsExcept for the mild-type, the levels of sCD138 in the moderate-, severe- and critical-type patients during the acute stage were significantly higher than that of the convalescent stage and the control (P<0.05). With the aggravation of the disease, the levels of sCD138 during the acute stage had an increasing tendency, while demonstrated no significant difference among the moderate-, severe- and critical-type patients (P>0.05). sCD138 was negatively correlated with Fib, PLT and ALB, and was positively correlated with WBC and AST (P<0.05). sCD138 demonstrated predictive effectiveness for prognosis with the area under the curve (AUC) of 0.778 (P<0.001).ConclusionDynamic detection of plasma sCD138 might be benefit to evaluating the disease severity and prognosis of the patients with HFRS.

Impact Coatings fuel cell coatings for Michelin, China deal

Swedish-based Impact Coatings, which develops and sells advanced technology for physical vapour deposition (PVD) coatings of fuel cell plates and vehicle reflectors, has received its first production order for Ceramic MaxPhase™ coatings from Michelin. Impact Coatings has also signed a cooperation agreement with Telos Auto Power Systems in China, to develop advanced fuel cell systems utilising Impact Coatings' bipolar plate coating technology.

Research progress of the mechanisms underlying transition of acute systemic inflammation to central inflammation

Background Systemic and central inflammation play important roles in surgery, trauma, and infection. It is still unclear how acute systemic inflammation develops into central inflammation. Objective To review the potential mechanisms and substrates for the transition from acute systemic inflammation to central inflammation in order to provide insights for preventing or alleviating postoperative neuroinflammation and cognitive dysfunction. Content Inflammation in the central nervous system is commonly seen in surgeries in non-nervous systems, septicemia, burn, and injury of organs, and might incur cognitive dysfunctions and neurodegenerative diseases. Inflammation factors and cytokines released during systemic inflammation increase permeability of blood-brain-brarrier (BBB), resulting in infiltration of inflammation factors, cytokines, and immune cells into the brain. The increase of permeability of blood-brain-brarrier is related to the damage of the integrity of endothelia glycocalyx layer following sepsis, inflammation, and dramatic hemodynamic perturbation, as well. Trend To improve integrity of endothelia glycocalyx layer, maintaining normal permeability of blood-brain-brarrier, may be a strategy to prevent the transition of systemic inflammation to central inflammation and neurodegenerative diseases. Key words: Systemic inflammation; Central inflammation; Lipopolysaccharide; Inflammatory factor; Endothelial surface layer

Large-Area Self-Assembly of Silica Microspheres/Nanospheres by Temperature-Assisted Dip-Coating.

This work reports a temperature-assisted dip-coating method for self-assembly of silica (SiO2) microspheres/nanospheres (SPs) as monolayers over large areas (∼cm2). The area over which self-assembled monolayers (SAMs) are formed can be controlled by tuning the suspension temperature (Ts), which allows precise control over the meniscus shape. Furthermore, the formation of periodic stripes of SAMs, with excellent dimensional control (stripe width and stripe-to-stripe spacing), is demonstrated using a suitable set of dip-coating parameters. These findings establish the role of Ts, and other parameters such as withdrawal speed (Vw), withdrawal angle (θw), and withdrawal step length (Lw). For Ts ranged between 25 and 80 °C, the morphological analysis of dip-coatings shows layered structures comprising of defective layers (25-60 °C), single layers (70 °C), and multilayers (>70 °C) owing to the variation of SP flux at the meniscus/substrate assembling interface. At Ts = 70 °C, there is an optimum Vw, approximately equal to the downshift speed of the meniscus (Vm = 1.3 μm/s), which allows the SAM formation over areas (2.25 cm2) roughly 10 times larger than reported in the literature using nanospheres. Finally, the large-area SAM is used to demonstrate the enhanced performance of antireflective coatings for photovoltaic cells and to create metal nanomesh for Si nanowire synthesis.

General and Facile Coating of Single Cells via Mild Reduction

Cell surface modification has been extensively studied to enhance the efficacy of cell therapy. Still, general accessibility and versatility are remaining challenges to meet the increasing demand for cell-based therapy. Herein, we present a facile and universal cell surface modification method that involves mild reduction of disulfide bonds in cell membrane protein to thiol groups. The reduced cells are successfully coated with biomolecules, polymers, and nanoparticles for an assortment of applications, including rapid cell assembly, in vivo cell monitoring, and localized cell-based drug delivery. No adverse effect on cellular morphology, viability, proliferation, and metabolism is observed. Furthermore, simultaneous coating with polyethylene glycol and dexamethasone-loaded nanoparticles facilitates enhanced cellular activities in mice, overcoming immune rejection.

The temporal response and mechanism of action of tranexamic acid in endothelial glycocalyx degradation.

The endothelial glycocalyx (GCX) plays an important role in vascular barrier function. Damage to the GCX occurs due to a variety of causes including hypoxia, ischemia-reperfusion, stress-related sympathoadrenal activation, and inflammation. Tranexamic acid (TXA) may prevent GCX degradation. The therapeutic window for TXA administration and the mechanism of action has been under review. Membrane-anchored proteases (sheddases) are key components in endothelial cell biology including the regulation of vascular permeability. The effect of TXA administration on stress-related GCX damage, and the role of sheddases in this process was studied in a cell-based model. Confluent human umbilical vein endothelial cells (HUVEC) were exposed to hydrogen peroxide and/or epinephrine (EPI) to stimulate postshock reperfusion. TXA was added at various times after hydrogen peroxide (H2O2) and/or EPI exposure. GCX degradation was indexed by syndecan-1 and hyaluronic acid release. Activation of endothelial sheddases was indexed by A Disintegrin and Metalloproteinase-17 and matrix metalloproteinase-9 activity in culture supernatants. Exposure of HUVEC to either/both EPI and H2O2 resulted in a cellular stress and GCX disruption demonstrated by increased levels of syndecan-1 shedding, hyaluronic acid release, tumor necrosis factor-α release. Shedding of these GCX components was associated with increased activity of both A Disintegrin and Metalloproteinase-17 and matrix metalloproteinase. Disruption of the GCX was further demonstrated via fluorescent imaging, which demonstrated disruption after exposure to either/both H2O2 and EPI. Early administration of either TXA or doxycycline resulted in preservation of the GCX. Late administration of TXA had no effect, whereas doxycycline had some residual protective effect. Tranexamic acid as a serine protease inhibitor prevented GCX degradation via inhibition of endothelial sheddase activation. This effect was not apparent when TXA was administered greater than 60 minutes after "simulated" reperfusion. Our study supports the clinical practice of early TXA administration in the severely injured patient.

Development of the damaged glycocalyx hypothesis - A review

Development of the damaged glycocalyx hypothesis - A review

A Novel Therapeutic Strategy for Pancreatic Cancer: Targeting Cell Surface Glycan Using rBC2LC-N Lectin-Drug Conjugate (LDC).

Various cancers, including pancreatic ductal adenocarcinoma (PDAC), remain intractable even with costly tumor-targeting antibody drugs. Because the outermost coatings of cancer cells are composed of cell-specific glycan layers (glycocalyx), lectins, proteins with glycan-binding potential, were evaluated for possible use as drug carriers in PDAC treatment. A human PDAC cell line with well-to-moderately differentiated properties (Capan-1) was subjected to lectin microarray analysis to identify specific lectin-glycan pairs. The selected lectin was fused with a bacterial exotoxin for the construction of a lectin-drug conjugate (LDC), and its safety and antitumor effects were evaluated. A specific affinity between a recombinant bacterial C-type lectin (rBC2LC-N) and Capan-1 was identified, and its positivity was confirmed in 69 human samples. In contrast to the belief that all lectins mediate harmful hemagglutination, rBC2LC-N did not cause hemagglutination with human erythrocytes and was safely administered to mice. The 50% inhibitory concentration of LDC to Capan-1 (1.04 pg/mL = 0.0195 pmol/L) was 1/1,000 lower than that reported for conventional immunotoxins. The intraperitoneal administration of LDC reduced the tumor weight from 390 to 130.8 mg (P < 0.01) in an orthotopic model and reduced the number of nodules from 48 to 3 (P < 0.001) and improved survival from 62 to 105 days in a peritoneal dissemination model (P < 0.0001). In addition, the effect of LDC was reproduced in nodules from patient-derived PDAC xenografts through intravenous injection. Herein, we show the concept of utilizing lectins as drug carriers to target glycans on the cancer cell surface, highlighting new insights into cancer treatments. Mol Cancer Ther; 17(1); 183-95. ©2017 AACR.

Effect of substrate coating material on spontaneous activity of human-induced pluripotent stem cell-derived neuronal stem cells

Effect of substrate coating material on spontaneous activity of human-induced pluripotent stem cell-derived neuronal stem cells

High DNA stability in white blood cells and buffy coat lysates stored at ambient temperature under anoxic and anhydrous atmosphere.

Conventional storage of blood-derived fractions relies on cold. However, lately, ambient temperature preservation has been evaluated by several independent institutions that see economic and logistic advantages in getting rid of the cold chain. Here we validated a novel procedure for ambient temperature preservation of DNA in white blood cell and buffy coat lysates based on the confinement of the desiccated biospecimens under anoxic and anhydrous atmosphere in original hermetic minicapsules. For this validation we stored encapsulated samples either at ambient temperature or at several elevated temperatures to accelerate aging. We found that DNA extracted from stored samples was of good quality with a yield of extraction as expected. Degradation rates were estimated from the average fragment size of denatured DNA run on agarose gels and from qPCR reactions. At ambient temperature, these rates were too low to be measured but the degradation rate dependence on temperature followed Arrhenius’ law, making it possible to extrapolate degradation rates at 25°C. According to these values, the DNA stored in the encapsulated blood products would remain larger than 20 kb after one century at ambient temperature. At last, qPCR experiments demonstrated the compatibility of extracted DNA with routine DNA downstream analyses. Altogether, these results showed that this novel storage method provides an adequate environment for ambient temperature long term storage of high molecular weight DNA in dehydrated lysates of white blood cells and buffy coats.

Glycans in Infectious Diseases. A Molecular Recognition Perspective.

From the simplest bacteria to the highest complex mammals, including humans, every single cell is covered by a dense coat of glycans. Glycans are involved in almost every biological process that takes place in our body, playing a central role in the communication between cells and their environment. Glycans are also involved in infectious diseases, which arise from the specific interaction between glycans of the pathogen cell coat and specific receptors on the host cell or vice versa. The understanding of the mechanisms governing these specific carbohydrateprotein interactions, at atomic and molecular levels, is crucial to develop new drugs able to block the infection and to avoid the disease. Recent advances in biophysical techniques allow for a complete picture of the hostpathogen infection event, unveiling the key aspects of the molecular interaction and, thus, providing an opportunity to interfere with it. In this general review, we discuss some recent contributions, providing a summary of what we consider the most innovative and inspiring research lines to the field.

Experimental and Numerical Study of the Effect of Gas-Shrouded Plasma Spraying on Cathode Coating of Alkaline Electrolysis Cells

The aim of this work is to improve the performance of electrodes prepared via atmospheric plasma spray by means of gas shrouding which is expected to apparently reduce the oxygen content of the plasma plume and subsequently improve the coating quality. Electrodes with dual-layer coating for alkaline water electrolysis were deposited on Ni-coated perforated substrates. Microstructure and morphology were studied by SEM. Element content was measured by EDS. Enthalpy probe was employed for measuring plasma temperature and velocity as well as the gas composition. For verifying and better understanding the shrouding effect numerical calculation was carried out according to the experimental settings. Electrochemical test was carried out to validate the shrouding effect. The results showed slight protecting effect of gas shrouding on plasma plume and the final coating. Over the dual-layer section, the measured oxygen fraction was 3.46 and 3.15% for the case without gas shrouding and with gas shrouding, respectively. With gas shrouding the coating exhibited similar element contents as the coating sprayed by VPS, while no obvious improvement was observed in the microstructure or the morphology. Evident electrochemical improvement was nevertheless achieved that with gas shrouding the electrode exhibited similar performance as that of the VPS-sprayed electrode.

Single-Molecule Imaging of Proteoglycans in the Pericellular Matrix

The pericellular matrix is a robust, hyaluronan-rich polymer brush-like structure that controls access to the cell surface, and plays an important role in cell adhesion, migration, and proliferation. We report the observation of single bottlebrush proteoglycan dynamics in the pericellular matrix of living chondrocytes. Our investigations show that the pericellular matrix undergoes gross extension on the addition of exogenous aggrecan, and that this extension is significantly in excess of that observed in traditional particle exclusion assays. The mean-square displacement of single, bound proteoglycans increases with distance to cell surface, indicating reduced confinement by neighboring hyaluronan-aggrecan complexes. This is consistent with published data from quantitative particle exclusion assays that show openings in the pericellular matrix microstructure ranging from ∼150 nm near the cell surface to ∼400 nm near the cell edge. In addition, the mobility of tethered aggrecan drops significantly when the cell coat is enriched with bottlebrush proteoglycans. Single-molecule imaging in this thick polysaccharide matrix on living cells has significant promise in the drive to elucidate the role of the pericellular coat in human health.

First-Principles Study of Pressure Dependence of Optical Spectra of MnS

MnS is a semiconductor material that can be used in solar cell coatings as window/buffer material and in many other applications. The present contribution deals with the optical properties of MnS in the zinc-blende structure using ab initio calculations within the generalized gradient approximation. Features such as dielectric function, refractive index, reflectivity, optical absorption, conductivity, and electron loss function spectra have been reported and their pressure dependence has been examined and discussed. The information gathered from the present study can be useful for photovoltaic applications.

Regeneration of glycocalyx by heparan sulfate and sphingosine 1-phosphate restores inter-endothelial communication.

Vasculoprotective endothelium glycocalyx (GCX) shedding plays a critical role in vascular disease. Previous work demonstrated that GCX degradation disrupts endothelial cell (EC) gap junction connexin (Cx) proteins, likely blocking interendothelial molecular transport that maintains EC and vascular tissue homeostasis to resist disease. Here, we focused on GCX regeneration and tested the hypothesis that vasculoprotective EC function can be stimulated via replacement of GCX when it is shed. We used EC with [i] intact heparan sulfate (HS), the most abundant GCX component; [ii] degraded HS; or [iii] HS that was restored after enzyme degradation, by cellular self-recovery or artificially. Artificial HS restoration was achieved via treatment with exogenous HS, with or without the GCX regenerator and protector sphingosine 1- phosphate (S1P). In these cells we immunocytochemically examined expression of Cx isotype 43 (Cx43) at EC borders and characterized Cx-containing gap junction activity by measuring interendothelial spread of gap junction permeable Lucifer Yellow dye. With intact HS, 60% of EC borders expressed Cx43 and dye spread to 2.88 ± 0.09 neighboring cells. HS degradation decreased Cx43 expression to 30% and reduced dye spread to 1.87± 0.06 cells. Cellular self-recovery of HS restored baseline levels of Cx43 and dye transfer. Artificial HS recovery with exogenous HS partially restored Cx43 expression to 46% and yielded dye spread to only 1.03 ± 0.07 cells. Treatment with both HS and S1P, recovered HS and restored Cx43 to 56% with significant dye transfer to 3.96 ± 0.23 cells. This is the first evidence of GCX regeneration in a manner that effectively restores vasculoprotective EC communication.

AC/DC electrical conduction and dielectric properties of PMMA/PVAc/C60 down-shifting nanocomposite films

Polymer nanocomposite films were prepared by doping fullerene C60 in polymer blend composed of polymethacrylate/polyvinyl acetate blends (PMMA/PVAc) using solution cast technique. The films were characterized by differential scanning calorimeter (DSC), Transmission electron microscope (TEM), DC/AC electrical conductivity and dielectric measurements in the frequency range (100 Hz- 1 MHz). The glass transition temperature, Tg, was increased by increasing the concentration of fullerene C60; this property reflects the increase of thermal stability by increasing the nanofiller content. The DC and AC electrical conductivities were enhanced by increasing C60 concentration due to the electron hopping or tunneling between filled and empty localized states above Tg. The relaxation time was determined from the αβ -relaxations and found to be attenuated by increasing the temperature as a typical behavior of amorphous polymers. The calculated values of thermodynamic parameters revealed the increase of molecular stability by increasing the doping concentration; this feature supports the application of PMMA/PVAc/C60 nanocomposite films in a wide scale of solar energy conversion applications such as luminescent down-shifting (LDS) coatings for photovoltaic cells.