Glycocalyx Structure Research Articles

The endothelial glycocalyx: research methods and prospects for their use in endothelial dysfunction assessment

A modern concept of the endothelial dysfunction in the pathogenesis of many diseases includes the glycocalyx damage along with impaired of the morphology and function of endothelial cells. The glycocalyx is a gel-like submembrane complex of protein-carbohydrate, carbohydrate-lipid components and plasma molecules forming a three-dimensional network on the luminal surface of the endothelium. The features of the borderline location of endothelial glycocalyx in the vascular system determine various important functions: barrier, regulatory, anti-inflammatory, antithrombotic, mechanotransduction ones, etc. In recent years, due to the improvement of visualization methods endothelial glycocalyx structure has been detailed. Its physiological functions and the role in the development of some pathological conditions have been clarified. Modern methods of glycocalyx assessment include invasive and non-invasive research techniques. The most promising ones are dark-field microscopy and the determination of glycocalyx-associated biochemical markers. Endothelial glycocalyx damage is a universal pathogenetic component and the earliest marker of the development of most diseases. Therefore, the endothelial glycocalyx assessment refers to promising areas of research. The ability to correlate with other prognostic indicators allows us to consider the endothelial glycocalyx damage as a marker of the poor health prognosis. That is why, assessment of the endothelial glycocalyx condition will allow to personalize treatment and to prevent the diseases progression.

Flow-regulated endothelial glycocalyx determines metastatic cancer cell activity.

Cancer metastasis and secondary tumor initiation largely depend on circulating tumor cell (CTC) and vascular endothelial cell (EC) interactions by incompletely understood mechanisms. Endothelial glycocalyx (GCX) dysfunction may play a significant role in this process. GCX structure depends on vascular flow patterns, which are irregular in tumor environments. This work presents evidence that disturbed flow (DF) induces GCX degradation, leading to CTC homing to the endothelium, a first step in secondary tumor formation. A 2-fold greater attachment of CTCs to human ECs was found to occur under DF conditions, compared to uniform flow (UF) conditions. These results corresponded to an approximately 50% decrease in wheat germ agglutinin (WGA)-labeled components of the GCX under DF conditions, vs UF conditions, with undifferentiated levels of CTC-recruiting E-selectin under DF vs UF conditions. Confirming the role of the GCX, neuraminidase induced the degradation of WGA-labeled GCX under UF cell culture conditions or in Balb/C miceand led to an over 2-fold increase in CTC attachment to ECs or Balb/C mouse lungs, respectively, compared to untreated conditions. These experiments confirm that flow-induced GCX degradation can enable metastatic CTC arrest. This work, therefore, provides new insight into pathways of secondary tumor formation.

The endothelial glycocalyx: Structure and function in health and critical illness.

To conduct a narrative review of the current literature in reference to the structure and function of the endothelial glycocalyx (EG) and its contribution to the pathophysiology of conditions relevant to the veterinary emergency and critical care clinician. Novel therapies for restoring or preserving the EG will also be discussed. Online databases (PubMed, CAB abstracts, Scopus) were searched between January 1st 2017 and May 1st 2017 for English language articles without publication date restriction. Keywords included EG, endothelial surface layer, degradation, syndecan-1, heparan sulfate, critical illness, sepsis, trauma, and therapeutics. The EG is a complex and important structure located on the luminal surface of all blood vessels throughout the body. It plays an important role in normal vascular homeostasis including control of fluid exchange across the vascular barrier. Loss or degradation of the EG has an impact on inflammation, coagulation, and vascular permeability and tone. These changes are essential components in the pathophysiology of many conditions including sepsis and trauma. A substantial body of experimental animal and human clinical research over the last decade has demonstrated increased circulating concentrations of EG degradation products in these conditions. However, veterinary-specific research into the EG and critical illness is currently lacking. The utility of EG degradation products as diagnostic and prognostic tools continues to be investigated and new therapies to preserve or improve EG structure and function are under development. The recognition of the presence of the EG has changed our understanding of transvascular fluid flux and the pathophysiology of many conditions of critical illness. The EG is an exciting target for novel therapeutics to improve morbidity and mortality in conditions such as sepsis and trauma.

Shear Stress Regulation of Endothelial Glycocalyx Structure Is Determined by Glucobiosynthesis.

Endothelial cells exposed to laminar shear stress express a thick glycocalyx on their surface that plays an important role in reducing vascular permeability and endothelial anti-inflammatory, antithrombotic, and antiangiogenic properties. Production and maintenance of this glycocalyx layer is dependent on cellular carbohydrate synthesis, but its regulation is still unknown. Approach and Results: Here, we show that biosynthesis of the major structural component of the endothelial glycocalyx, hyaluronan, is regulated by shear. Both in vitro as well as in in vivo, hyaluronan expression on the endothelial surface is increased on laminar shear and reduced when exposed to oscillatory flow, which is regulated by KLF2 (Krüppel-like Factor 2). Using a CRISPR-CAS9 edited small tetracysteine tag to endogenous HAS2 (hyaluronan synthase 2), we demonstrated increased translocation of HAS2 to the endothelial cell membrane during laminar shear. Hyaluronan production by HAS2 was shown to be further driven by availability of the hyaluronan substrates UDP-glucosamine and UDP-glucuronic acid. KLF2 inhibits endothelial glycolysis and allows for glucose intermediates to shuttle into the hexosamine- and glucuronic acid biosynthesis pathways, as measured using nuclear magnetic resonance analysis in combination with 13C-labeled glucose. These data demonstrate how endothelial glycocalyx function and functional adaptation to shear is coupled to KLF2-mediated regulation of endothelial glycolysis.

Sphingosine-1-Phosphate Reduces Hemorrhagic Shock and Resuscitation-Induced Microvascular Leakage by Protecting Endothelial Mitochondrial Integrity.

Excessive microvascular permeability is a serious complication following hemorrhagic shock and resuscitation (HSR). S1P has been shown to ameliorate microvascular leakage in a model of combined alcohol intoxication and HSR. In the current study, we tested the hypothesis that S1P reduces HSR-induced microvascular leakage by preserving endothelial cell junctional structure and the endothelial glycocalyx through the protection of mitochondrial function. We used an established in vivo rat model of conscious HSR and assessed microvascular leakage, endothelial glycocalyx integrity, and mitochondrial function by intravital microscopy. Junctional integrity in the mesenteric microcirculation was assessed by confocal microscopy. Cultured rat intestinal microvascular endothelial cells monolayers were used to test the ability of S1P to protect against glycocalyx shedding and endothelial barrier dysfunction caused by direct disruption of mitochondrial integrity due to inhibition of mitochondrial complex III. The results show that in vivo, S1P protects against HSR-induced hyperpermeability, preserves the expression of adherens junctional proteins, and protects against glycocalyx degradation. S1P treatment during HSR also protects against mitochondrial membrane depolarization. S1P also protects against mitochondrial dysfunction-induced endothelial barrier dysfunction and glycocalyx degradation by acting through mitochondrial complex III. Taken together, our data indicate that S1P protects against HSR-induced mitochondrial dysfunction in endothelial cells, which in turn improves the structure of the endothelial glycocalyx after HSR and allows for better junctional integrity to the prevention of excess microvascular permeability.

Glomerular Function and Structural Integrity Depend on Hyaluronan Synthesis by Glomerular Endothelium.

A glycocalyx envelope consisting of proteoglycans and adhering proteins covers endothelial cells, both the luminal and abluminal surface. We previously demonstrated that short-term loss of integrity of the luminal glycocalyx layer resulted in perturbed glomerular filtration barrier function. To explore the role of the glycocalyx layer of the endothelial extracellular matrix in renal function, we generated mice with an endothelium-specific and inducible deletion of hyaluronan synthase 2 (Has2), the enzyme that produces hyaluronan, the main structural component of the endothelial glycocalyx layer. We also investigated the presence of endothelial hyaluronan in human kidney tissue from patients with varying degrees of diabetic nephropathy. Endothelial deletion of Has2 in adult mice led to substantial loss of the glycocalyx structure, and analysis of their kidneys and kidney function showed vascular destabilization, characterized by mesangiolysis, capillary ballooning, and albuminuria. This process develops over time into glomerular capillary rarefaction and glomerulosclerosis, recapitulating the phenotype of progressive human diabetic nephropathy. Using a hyaluronan-specific probe, we found loss of glomerular endothelial hyaluronan in association with lesion formation in tissue from patients with diabetic nephropathy. We also demonstrated that loss of hyaluronan, which harbors a specific binding site for angiopoietin and a key regulator of endothelial quiescence and maintenance of EC barrier function results in disturbed angiopoietin 1 Tie2. Endothelial loss of hyaluronan results in disturbed glomerular endothelial stabilization. Glomerular endothelial hyaluronan is a previously unrecognized key component of the extracelluar matrix that is required for glomerular structure and function and lost in diabetic nephropathy.

Preparation of Small-Diameter Tissue-Engineered Vascular Grafts Electrospun from Heparin End-Capped PCL and Evaluation in a Rabbit Carotid Artery Replacement Model.

Aiming to construct small diameter (ID <6 mm) off-the-shelf tissue-engineered vascular grafts, the end-group heparinizd poly(ε-caprolactone) (PCL) is synthesized by a three-step process and then electrospun into an inner layer of double-layer vascular scaffolds (DLVSs) showing a hierarchical double distribution of nano- and microfibers. Afterward, PCL without the end-group heparinization is electrospun into an outer layer. A steady release of grafted heparin and the existence of a glycocalyx structure give the grafts anticoagulation activity and the conjugation of heparin also improves hydrophilicity and accelerates degradation of the scaffolds. The DLVSs are evaluated in six rabbits via a carotid artery interpositional model for a period of three months. All the grafts are patent until explantation, and meanwhile smooth endothelialization and fine revascularization are observed in the grafts. The composition of the outer layer of scaffolds exhibits a significant effect on the aneurysm dilation after implantation. Only one aneurysm dilation is detected at two months and no calcification is formed in the follow-up term. How to prevent aneurysms remains a challenging topic.

Mechanotransmission of haemodynamic forces by the endothelial glycocalyx in a full-scale arterial model.

The glycocalyx has been identified as a key mechano-sensor of the shear forces exerted by streaming blood onto the vascular endothelial lining. Although the biochemical reaction to the blood flow has been extensively studied, the mechanism of transmission of the haemodynamic shear forces to the endothelial transmembrane anchoring structures and, consequently, to the subcellular elements in the cytoskeleton, is still not fully understood. Here we apply a multiscale approach to elucidate how haemodynamic shear forces are transmitted to the transmembrane anchors of endothelial cells. Wall shear stress time histories, as obtained from image-based computational haemodynamics models of a carotid bifurcation, are used as a load and a continuum model is applied to obtain the mechanical response of the glycocalyx all along the cardiac cycle. The main findings of this in silico study are that: (1) the forces transmitted to the transmembrane anchors are in the range of 1–10 pN, which is in the order of magnitude reported for the different conformational states of transmembrane mechanotranductors; (2) locally, the forces transmitted to the anchors of the glycocalyx structure can be markedly different from the near-wall haemodynamic shear forces both in amplitude and frequency content. The findings of this in silico approach warrant future studies focusing on the actual forces transmitted to the transmembrane mechanotransductors, which might outperform haemodynamic descriptors of disturbed shear as localizing factors of vascular disease.

Sphingosine‐1‐phosphate Enhances Mitochondrial Integrity and Endothelial Barrier Function Following Hemorrhagic Shock

Excessive microvascular permeability is a serious complication involved in traumatic injury and inflammatory diseases. Sphingosine‐1‐phosphate (S1P) has known endothelial barrier‐protective properties, and has been shown to ameliorate microvascular leakage in a model of combined alcohol intoxication and hemorrhagic shock and resuscitation (HSR). However, the mechanisms of S1P‐induced barrier protection during HSR are unknown. In the current study, we tested the hypothesis that S1P could enhance endothelial barrier function during hemorrhagic shock by preserving the integrity of junction proteins and the endothelial glycocalyx, and protecting mitochondrial function. We used an established in vivo rat model of conscious HSR and assessed microvascular leakage, endothelial glycocalyx integrity, and mitochondrial function by intravital microscopy. Assessment of adherens junction protein localization was done by immunofluorescence confocal microscopy after HSR. Cultured rat intestinal microvascular endothelial cell (RIMEC) monolayers were used to test the ability of S1P to protect against glycocalyx shedding and endothelial barrier dysfunction caused by direct disruption of mitochondrial integrity due to inhibition of mitochondrial complex III. Our in vivo results show that S1P protects against HSR‐induced hyperpermeability, preserves the expression of adherens junctional proteins, and protects against glycocalyx degradation. S1P treatment during HSR also protects against mitochondrial membrane depolarization. Besides that, S1P protects RIMECs against mitochondrial dysfunction‐induced endothelial barrier dysfunction and glycocalyx degradation by blocking complex III inhibition. Our results show that S1P protects against HSR‐induced mitochondrial dysfunction in endothelial cells, which in turn improves the structure of the endothelial glycocalyx after HSR and allows for better junctional integrity, preventing excess microvascular permeability.Support or Funding InformationSupported by NIH grants R01HL098215, R01GM120774, R01HL070752 and R01GM097270.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Equilibrium Modeling of the Mechanics and Structure of the Cancer Glycocalyx

The glycocalyx is a thick coat of proteins and carbohydrates on the outer surface of all eukaryotic cells. Overproduction of large, flexible or rod-like biopolymers, including hyaluronic acid and mucins, in the glycocalyx strongly correlates with the aggression of many cancer types. However, theoretical frameworks to predict the effects of these changes on cancer cell adhesion and other biophysical processes remain limited. Here, we propose a detailed modeling framework for the glycocalyx incorporating important physical effects of biopolymer flexibility, excluded volume, counterion mobility, and coupled membrane deformations. Because mucin and hyaluronic biopolymers are proposed to extend and rigidify depending on the extent of their decoration with side chains, we propose and consider two limiting cases for the structural elements of the glycocalyx: stiff beams and flexible chains. Simulations predict the mechanical response of the glycocalyx to compressive loads, which are imposed on cells residing in the highly confined spaces of the solid tumor or invaded tissues. Notably, the shape of the mechanical response transitions from hyperbolic to sigmoidal for more rod-like glycocalyx elements. These mechanical responses, along with the corresponding equilibrium protein organizations and membrane topographies, are summarized to aid in hypothesis generation and the evaluation of future experimental measurements. Overall, the modeling framework developed provides a theoretical basis for understanding the physical biology of the glycocalyx in human health.

Ultrasmall gold nanorods: synthesis and glycocalyx-related permeability in human endothelial cells.

BackgroundClinical data show shed endothelial glycocalyx (GCX) components in blood samples of atherosclerotic patients, linking atherosclerotic development to endothelial GCX integrity. Healthy GCX has pores no >7 nm, and shed GCX has even larger pores. Therefore, we suggest targeting and treating atherosclerosis-prone blood vessels by using nanoscale vehicles to deliver drugs via the nanoscale GCX as it becomes dysfunctional.Materials and methodsTo test our idea, we investigated permeability of nanoparticles in endothelium, as related to a GCX expression. The present work involves nanorods, which are expected to interact with larger portions of endothelial cell (EC) membranes, due to surface area of the nanorod long axis. Conventional nanorod diameters are orders of magnitude larger than the GCX pore size, so we adapted conventional synthesis methods to fabricate ultrasmall gold nanorods (GNRs). Our ultrasmall GNRs have an aspect ratio of 3.4, with a length of 27.9±3.1 nm and a diameter of 8.2±1.4 nm. In addition, we produced GNRs that are biocompatible and fluorescently visible, by coating the surface with functionalized polyethylene glycol and Alexa Fluor 647. To study GNR–GCX interactions, we used human ECs, for species relevance.ResultsUnder life-like flow conditions, the human ECs are densely covered with a 1.3 µm thick layer of GCX, which coincides with minimal GNR permeability. When the GCX is weakened from lack of flow (static culture) or the presence of GCX degradation enzyme in the flow stream, the GCX shows 40% and 60% decreased thickness, respectively. GCX weakness due to lack of flow only slightly increases cellular permeability to GNRs, while GCX weakness due to the presence of enzyme in the flow leads to substantial increase in GNR permeability.ConclusionThese results clarify that the GCX structure is an avenue through which drug-carrying nanoparticles can be delivered for targeting affected blood vessels to treat atherosclerosis.

ВПЛИВ ЕКСТРАКТІВ І СУСПЕНЗІЙ ЯКОНА (SMALLANTHUS SONCHIFOLIA) НА ЗМІНИ СТРУКТУРИ ВУГЛЕВОДНИХ ДЕТЕРМІНАНТ ГЛІКОКОН’ЮГАТІВ МЕМБРАН ЕРИТРОЦИТІВ ЗА УМОВ ЕКСПЕРИМЕНТАЛЬНОГО ЦУКРОВОГО ДІАБЕТУ

The article presents the results of the study dedicated to the influence of yacons leaves and root tubers water extracts and suspensions of roots in a dose of 500 mg / kg on changes in the structure of erythrocytes membranes glycoconjugates carbohydrate determinants and erythrocytes resistance to acidic hemolytic activity under conditions of experimental diabetes mellitus (EDM) type 1. Investigated pathology is accompanied by a violation of the functional state and by a change in the glycane profile of the erythrocyte plasma membrane. In particular, it has been established that the development of diabetes mellitus is accompanied by a decrease in the stability of erythrocytes membranes to the action of acid hemolysis, an increase in the number of physiologically old erythrocytes and a decrease in the content of sialic acids in oligosaccharide glycoconjugates of erythrocyte membranes attached by (α2→6)- and (α2→3)- glycoside bonds to subterminal sugars, as well as the growth of the expression of subterminal galactose residues. At administration of the studied extracts and suspensions to animals with an EDM the positive effect on the physicochemical state of the erythrocyte membranes was noted. The use of yacons extracts and suspensions cause in bloodstream the growth of number of erythrocytes young forms and middle-aged cells, on the background lowering of red blood cells old forms. It was established that the most pronounced effect is inherent in water suspensions of yacons roots tubers, the application of which leads to the normalization of the structure of the membranes glycocalyx of red blood cells, to normalization of sialic acids content on the surface of erythrocytes, with the simultaneous decrease in the content of subterminal galactose residues. Corrective effect of investigated extracts and suspensions on the physicochemical state of erythrocyte membranes can be an experimental confirmation of the need for the development of functional foods based on yacon, which can be used in complex therapy of diabetes and prevent the harmful effects of hyperglycemia.

Brain-Specific Ultrastructure of Capillary Endothelial Glycocalyx and Its Possible Contribution for Blood Brain Barrier

Endothelial glycocalyx coats healthy vascular endothelium and plays an important role in vascular homeostasis. Although cerebral capillaries are categorized as continuous, as are those in the heart and lung, they likely have specific features related to their function in the blood brain barrier. To test that idea, brains, hearts and lungs from C57BL6 mice were processed with lanthanum-containing alkaline fixative, which preserves the structure of glycocalyx, and examined using scanning and transmission electron microscopy. We found that endothelial glycocalyx is present over the entire luminal surface of cerebral capillaries. The percent area physically covered by glycocalyx within the lumen of cerebral capillaries was 40.1 ± 4.5%, which is significantly more than in cardiac and pulmonary capillaries (15.1 ± 3.7% and 3.7 ± 0.3%, respectively). Upon lipopolysaccharide-induced vascular injury, the endothelial glycocalyx was reduced within cerebral capillaries, but substantial amounts remained. By contrast, cardiac and pulmonary capillaries became nearly devoid of glycocalyx. These findings suggest the denser structure of glycocalyx in the brain is associated with endothelial protection and may be an important component of the blood brain barrier.

209. The microvascular endothelial glycocalyx: impaired barrier function in preeclampsia with small gestational age neonates

Introduction The glycocalyx, the endothelial lining of membrane-bound proteoglycans and glycoproteins, serves as a functional barrier between flowing red blood cells (RBC) and the endothelial cell membrane. Recent studies suggest that damage to the glycocalyx, which can be reflected by deeper radial penetration of RBC into the glycocalyx, contributes to microvascular dysfunction. Hypothesis We hypothesized that women with preeclampsia, especially those who deliver a small-for-gestational age neonate (PE + SGA, birthweight Methods We compared 30 women with a normotensive pregnancy (NP) to 30 women with preeclampsia (PE), matched for gestational age at time of analysis and presence/absence of labor. Using sidestream dark field imaging of the sublingual microcirculation, we measured 1) the accessibility of the RBC into the glycocalyx layer (perfused boundary region, PBR), defined as the difference between median and maximal RBC column widths, and 2) the microvascular density (total length of perfused microvessels/mm2 area). In healthy vessels, PBR is relatively small due to limited penetration of RBC into the glycocalyx. Results We observed no significant group differences in microvascular density (229 ± 75 mm/mm2 PE, 247 ± 88 mm/mm2 NP, P = 0.48) or PBR (2.20 ± 0.22 μm PE, 2.14 ± 0.30 μm NP, P = 0.14). However, the subset of women with PE + SGA (n = 12) evidenced a significant reduction in vascular density (205 ± 67 vs 306 ± 71 mm/mm2, P Discussion The increase in RBC encroachment (PBR) into the microvascular glycocalyx in PE + SGA compared to NP indicates a change in glycocalyx structure which may be associated with a reduced vascular protective function of the glycocalyx, possibly contributing to endothelial and microvascular dysfunction. Funded by NIH-NICHD (P01 HD030367, R21 HD083659), and AHA16SFRN27870000.

Luminescence from Lanthanide(III) Ions Bound to the Glycocalyx of Chinese Hamster Ovary Cells.

Lanthanide(III) ions bind to the glycocalyx of Chinese Hamster Ovary (CHO) cells and give rise to a unique luminescent fingerprint. Following direct excitation of terbium(III) and europium(III) ions in the visible part of the spectrum, we are able to collect emission spectra pixel-by-pixel in images of CHO cells. Following data analysis that removes the background signal, the fine structure of the europium(III) luminescence indicate that the lanthanide(III) ions are bound to a single structure of the CHO cell glycocalyx. This was deduced from the fact that the structure-sensitive emission spectrum of europium is unchanged throughout the investigated samples.

Oncotically Driven Control over Glycocalyx Dimension for Cell Surface Engineering and Protein Binding in the Longitudinal Direction

Here we present a simple technique for re-directing reactions on the cell surface to the outermost region of the glycocalyx. Macromolecular crowding with inert polymers was utilized to reversibly alter the accessibility of glycocalyx proteoglycans toward cell-surface reactive probes allowing for reactivity control in the longitudinal direction (‘z’-direction) on the glycocalyx. Studies in HUVECs demonstrated an oncotically driven collapse of the glycocalyx brush structure in the presence of crowders as the mechanism responsible for re-directing reactivity. This phenomenon is consistent across a variety of macromolecular agents including polymers, protein markers and antibodies which all displayed enhanced binding to the outermost surface of multiple cell types. We then demonstrated the biological significance of the technique by increasing the camouflage of red blood cell surface antigens via a crowding-enhanced attachment of voluminous polymers to the exterior of the glycocalyx. The accessibility to Rhesus D (RhD) and CD47 proteins on the cell surface was significantly decreased in crowding-assisted polymer grafting in comparison to non-crowded conditions. This strategy is expected to generate new tools for controlled glycocalyx engineering, probing the glycocalyx structure and function, and improving the development of cell based therapies.

The Role of Plasma Transfusion in Massive Bleeding: Protecting the Endothelial Glycocalyx?

Massive hemorrhage is a leading cause of death worldwide. During the last decade several retrospective and some prospective clinical studies have suggested a beneficial effect of early plasma-based resuscitation on survival in trauma patients. The underlying mechanisms are unknown but appear to involve the ability of plasma to preserve the endothelial glycocalyx. In this mini-review, we summarize current knowledge on glycocalyx structure and function, and present data describing the impact of hemorrhagic shock and resuscitation fluids on glycocalyx. Animal studies show that hemorrhagic shock leads to glycocalyx shedding, endothelial inflammatory changes, and vascular hyper-permeability. In these animal models, plasma administration preserves glycocalyx integrity and functions better than resuscitation with crystalloids or colloids. In addition, we briefly present data on the possible plasma components responsible for these effects. The endothelial glycocalyx is increasingly recognized as a critical component for the physiological vasculo-endothelial function, which is destroyed in hemorrhagic shock. Interventions for preserving an intact glycocalyx shall improve survival of trauma patients.

Regimes of Flow over Complex Structures of Endothelial Glycocalyx: A Molecular Dynamics Simulation Study

Flow patterns on surfaces grafted with complex structures play a pivotal role in many engineering and biomedical applications. In this research, large-scale molecular dynamics (MD) simulations are conducted to study the flow over complex surface structures of an endothelial glycocalyx layer. A detailed structure of glycocalyx has been adopted and the flow/glycocalyx system comprises about 5,800,000 atoms. Four cases involving varying external forces and modified glycocalyx configurations are constructed to reveal intricate fluid behaviour. Flow profiles including temporal evolutions and spatial distributions of velocity are illustrated. Moreover, streamline length and vorticity distributions under the four scenarios are compared and discussed to elucidate the effects of external forces and glycocalyx configurations on flow patterns. Results show that sugar chain configurations affect streamline length distributions but their impact on vorticity distributions is statistically insignificant, whilst the influence of the external forces on both streamline length and vorticity distributions are trivial. Finally, a regime diagram for flow over complex surface structures is proposed to categorise flow patterns.

A Disintegrin and Metalloproteinase 15‐Mediated Glycocalyx Disruption Contributes to Vascular Leakage During Septic Injury

Disruption of vascular endothelial barrier results in excessive accumulation of plasma proteins and fluid within the interstitial space, exacerbating tissue injury caused by infection or sepsis. There is an urgent need to explore effective strategies to restore vascular barrier integrity in patients with sepsis. Glycocalyx, an endothelial surface coating matrix composed of glycoproteins and proteinglycans attached to glycoaminoglycan chains, has been shown to be protective of vascular barrier function. Degradation of glycocalyx has been implicated in the progression of septic injury, evidenced by increased serum levels of glycocalyx degradation products, including soluble CD44 and hyaluronic acid in sepsis patients, correlating with severity of injury. Currently, the molecular mechanisms underlying glycocalyx degradation are poorly understood. Here, we investigated whether and how this process involves a disintegrin and metalloproteinase 15 (ADAM15), a transmembrane sheddase capable of cleaving the ectodomains of membrane‐bounded proteins. Based on intravital microscopic measurements of dextran (150 kD) exclusion, we comparatively assessed glycocalyx thickness in wild‐type and ADAM15 knockout mice subjected to septic insult via lipopolysaccharides (LPS) injection. Our results indicated that LPS treatment induced a remarkable reduction in glycocalyx thickness in wide type mice, a response significantly attenuated in ADAM15−/− mice, suggesting the critical role of ADAM15 in LPS‐induced glycocalyx injury. Moreover, glycocalyx in mouse pulmonary microvasculature was observed by electron microscopy. Consistently, ADAM15 deficiency prevented the loss of glycocalyx coverage on the endothelial surface in pulmonary vessels following LPS challenge. To elucidate the underlying molecular details, we performed an in vitro cleavage assay, which confirmed the ability of recombinant ADAM15 to cleave CD44, an important glycoprotein expressed in glycocalyx, in a dose‐dependent manner. Further proteomic analysis revealed that the cleavage site of CD44 fell within the membrane‐proximal region of its ectodomain. Functionally, incubating endothelial cells with CD44 ectodomain resulted in barrier dysfunction and adherens junction disorganization. In line with these in vitro findings, mice with impaired glycocalyx exhibited vascular hyperpermeability after LPS challenge, as indicated by increased Evans Blue‐bound albumin extravasation in the lungs as well as enhanced albumin leakage from mesenteric vessels. In contrast, microvessels from ADAM15−/− mice with well‐preserved glycocalyx structure displayed an attenuated permeability response. Taken together, our studies suggest a novel function of ADAM15 in mediating glycocalyx injury during septic challenge.Support or Funding InformationThis work was supported by the National Institutes of Health Grants GM097270 and HL070752.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Large-scale molecular dynamics simulation of flow under complex structure of endothelial glycocalyx

In this research, large-scale molecular dynamics (MD) simulations were conducted to study the fluid dynamics inside the endothelial glycocalyx layer. A work flowchart regarding constructing the flow/glycocalyx system, undertaking production simulation using the MD method and post-processing was proposed. Following the flowchart, physiological and accelerating flow cases were simulated to reveal velocity and shear stress distributions over the dendritic (tree-like) structure of the glycocalyx, thereby contributing to understanding of the influence of biomolecular complex structures on flow profiles. Besides, the selection of thermostat algorithm was discussed. Results have shown that when the forcing is below a critical value, the velocity fluctuates around a zero mean along the height in the presence of the dendritic glycocalyx. When the forcing is larger than a critical value, the bulk flow was accelerated excessively, departing from the typical physiological flow. Furthermore, distributions of shear stress magnitude among three sub-regions in the ectodomain indicate that shear stress is enhanced near the membrane surface but is impaired in the sugar-chain-rich region due to the flow regulation by sugar chains. Finally, comparisons of velocity evolutions under two widely used thermostats (Lowe-Andersen and Berendsen thermostats) imply that the Lowe-Andersen algorithm is a suitable thermostat for flow problems.