Endothelial Surface Glycocalyx Research Articles

Microvascular perfusion, perfused boundary region and glycocalyx shedding in patients with autosomal dominant polycystic kidney disease: results from the GlycoScore III study.

Vascular abnormalities and endothelial dysfunction are part of the spectrum of autosomal dominant polycystic kidney disease (ADPKD). The mechanisms behind these manifestations, including potential effects on the endothelial surface layer (ESL) and glycocalyx integrity, remain unknown. Forty-five ambulatory adult patients with ADPKD were enrolled in this prospective, observational, cross-sectional, single-centre study. Fifty-one healthy volunteers served as a control group. All participants underwent real-time microvascular perfusion measurements of the sublingual microcirculation using sidestream dark field imaging. After image acquisition, the perfused boundary region (PBR), an inverse parameter for red blood cell (RBC) penetration into the ESL, was automatically calculated. Microvascular perfusion was assessed by RBC filling and capillary density. Concentrations of circulating glycocalyx components were determined by enzyme-linked immunosorbent assay. ADPKD patients showed a significantly larger PBR compared with healthy controls (2.09±0.23µm versus 1.79±0.25µm; P<.001). This was accompanied by significantly lower RBC filling (70.4±5.0% versus 77.9±5.4%; P<.001) as well as a higher valid capillary density {318/mm2 [interquartile range (IQR) 269-380] versus 273/mm2 [230-327]; P=.007}. Significantly higher plasma concentrations of heparan sulphate (1625±807ng/ml versus 1329±316ng/ml; P=.034), hyaluronan (111ng/ml [IQR 79-132] versus 92ng/ml [82-98]; P=.042) and syndecan-1 were noted in ADPKD patients compared with healthy controls (35ng/ml [IQR 27-57] versus 29ng/ml [23-42]; P=.035). Dimensions and integrity of the ESL are impaired in ADPKD patients. Increased capillary density may be a compensatory mechanism for vascular dysfunction to ensure sufficient tissue perfusion and oxygenation.

Characterization of Lung Injury following Abrin Pulmonary Intoxication in Mice: Comparison to Ricin Poisoning.

Abrin is a highly toxic protein obtained from the seeds of the rosary pea plant Abrus precatorius, and it is closely related to ricin in terms of its structure and chemical properties. Both toxins inhibit ribosomal function, halt protein synthesis and lead to cellular death. The major clinical manifestations following pulmonary exposure to these toxins consist of severe lung inflammation and consequent respiratory insufficiency. Despite the high similarity between abrin and ricin in terms of disease progression, the ability to protect mice against these toxins by postexposure antibody-mediated treatment differs significantly, with a markedly higher level of protection achieved against abrin intoxication. In this study, we conducted an in-depth comparison between the kinetics of in vivo abrin and ricin intoxication in a murine model. The data demonstrated differential binding of abrin and ricin to the parenchymal cells of the lungs. Accordingly, toxin-mediated injury to the nonhematopoietic compartment was shown to be markedly lower in the case of abrin intoxication. Thus, profiling of alveolar epithelial cells demonstrated that although toxin-induced damage was restricted to alveolar epithelial type II cells following abrin intoxication, as previously reported for ricin, it was less pronounced. Furthermore, unlike following ricin intoxication, no direct damage was detected in the lung endothelial cell population following abrin exposure. Reduced impairment of intercellular junction molecules following abrin intoxication was detected as well. In contrast, similar damage to the endothelial surface glycocalyx layer was observed for the two toxins. We assume that the reduced damage to the lung stroma, which maintains a higher level of tissue integrity following pulmonary exposure to abrin compared to ricin, contributes to the high efficiency of the anti-abrin antibody treatment at late time points after exposure.

Biophysical Properties of Endothelial Mechanotransduction Mediated by Glycocalyx

Endothelial surface glycocalyx (ESG) is a carbohydrate‐rich layer found on the endothelial cell (EC) surface lining all blood vessels' inner lumen. Composed of membrane glycoproteins, glycosaminoglycans (GAGs), such as hyaluronan and heparan sulfate (HS), and proteoglycans, the ESG forms a bulky, gel‐like layer serving critical functions in blood flow mechanotransduction, endothelial permeability maintenance, leukocyte adhesion, and inflammation control. ESG is known for sensing shear stress of blood flow and transducing the mechanical signal into nitric oxide (NO) production. NO is an essential signaling molecule to regulate vascular tone. To date, the molecular pathways of ESG‐mediated mechanotransduction have not been completely clear.We utilize a custom‐built AFM with fluorescence imaging capabilities to vertically stretch the ESG, which applies a pulling force to specific ESG components on the surface of a single live mouse brain endothelial cell (bEnd.3) and quantify the cell's NO production in real‐time. NO production is quantified by the fluorescent dye DAF‐FM. AFM probe is coated with monoclonal antibodies against HS or glypican‐1 (a major core protein for HS) to exert pulling forces onto the HS or glypican‐1 on bEnd.3. To ensure that DAF intensity is a signal of NO production, we treated the cells with L‐NAME (an inhibitor of endothelial nitric oxide synthase). The DAF intensity was greatly reduced, which confirms that the DAF intensity is representing NO production.The AFM anti‐glypican‐1 coated probe showed an increase in DAF intensity, while the anti‐HS probe is shown to have a slight rise in DAF intensity. Since HS may attach to other proteins besides glypican, the data suggest that glypican‐1 is a more critical mechanotransducer. Our prior study indicated cation‐permeable, transient receptor potential (TRP) ion channels mediate stretch‐induced NO production. Probes were coated in glypican‐1 while the cells were exposed to Amiloride or SKF96365, blockers of TRPP2 and TRPC1 channels, respectively. After the Amiloride‐treated cells were mechanically stimulated, there was a reduction of NO production. However, the amount of NO output was still significant over the basal levels observed in cells. When the SKF96365‐treated cells were mechanically stimulated, production was inhibited entirely, thus, demonstrating TRPC1 channels play a vital role in allowing the Ca2+ into the cell to begin NO production cascade.These findings confirm the critical roles of HS, glypican‐1, and TRP channels on the rapid NO production resulting from mechanical signaling and have important implications for ESG‐related cardiovascular diseases such as diabetes, strokes, cardiac arrest, and atherosclerosis. It has been shown that intracellular Ca2+regulates eNOS activity. In future studies, we will measure the Ca2+intake mediated by TRP channels and delineate the interplay among mechanical stretch, TRP channel activation, Ca2+intake and NO production.

Heparan sulfate mimetic fucoidan restores the endothelial glycocalyx and protects against dysfunction induced by serum of COVID-19 patients in the intensive care unit

Accumulating evidence proves that endothelial dysfunction is involved in coronavirus disease 2019 (COVID-19) progression. We previously demonstrated that the endothelial surface glycocalyx has a critical role in maintenance of vascular integrity. Here, we hypothesised that serum factors of severe COVID-19 patients affect the glycocalyx and result in endothelial dysfunction.We included blood samples of 32 COVID-19 hospitalised patients at the Leiden University Medical Center, of which 26 were hospitalised in an intensive care unit (ICU) and six on a non-ICU hospital floor; 18 of the samples were obtained from convalescent patients 6 weeks after hospital discharge, and 12 from age-matched healthy donors (control) during the first period of the outbreak. First, we determined endothelial (angiopoietin 2 (ANG2)) and glycocalyx degradation (soluble thrombomodulin (sTM) and syndecan-1 (sSDC1)) markers in plasma.In the plasma of COVID-19 patients, circulating ANG2 and sTM were elevated in patients in the ICU. Primary lung microvascular endothelial cell (HPMEC) and human glomerular microvascular endothelial cell (GEnC) cultured in the presence of these sera led to endothelial cell glycocalyx degradation, barrier disruption, inflammation and increased coagulation on the endothelial surface, significantly different compared to healthy control and non-ICU patient sera. These changes could all be restored in the presence of fucoidan.In conclusion, our data highlight the link between endothelial glycocalyx degradation, barrier failure and induction of a procoagulant surface in COVID-19 patients in ICU which could be targeted earlier in disease by the presence of heparan sulfate mimetics.

Blood\u2013brain barrier dysfunction in l-ornithine induced acute pancreatitis in rats and the direct effect of l-ornithine on cultured brain endothelial cells

BackgroundIn severe acute pancreatitis (AP) the CNS is affected manifesting in neurological symptoms. Earlier research from our laboratory showed blood–brain barrier (BBB) permeability elevation in a taurocholate-induced AP model. Here we aimed to further explore BBB changes in AP using a different, non-invasive in vivo model induced by l-ornithine. Our goal was also to identify whether l-ornithine, a cationic amino acid, has a direct effect on brain endothelial cells in vitro contributing to the observed BBB changes.MethodsAP was induced in rats by the intraperitoneal administration of l-ornithine-HCl. Vessel permeability and the gene expression of the primary transporter of l-ornithine, cationic amino acid transporter-1 (Cat-1) in the brain cortex, pancreas, liver and lung were determined. Ultrastructural changes were followed by transmission electron microscopy. The direct effect of l-ornithine was tested on primary rat brain endothelial cells and a triple co-culture model of the BBB. Viability and barrier integrity, including permeability and TEER, nitrogen monoxide (NO) and reactive oxygen species (ROS) production and NF-κB translocation were measured. Fluorescent staining for claudin-5, occludin, ZO-1, β-catenin, cell adhesion molecules Icam-1 and Vcam-1 and mitochondria was performed. Cell surface charge was measured by laser Doppler velocimetry.ResultsIn the l-ornithine-induced AP model vessel permeability for fluorescein and Cat-1 expression levels were elevated in the brain cortex and pancreas. On the ultrastructural level surface glycocalyx and mitochondrial damage, tight junction and basal membrane alterations, and glial edema were observed. l-ornithine decreased cell impedance and elevated the BBB model permeability in vitro. Discontinuity in the surface glycocalyx labeling and immunostaining of junctional proteins, cytoplasmic redistribution of ZO-1 and β-catenin, and elevation of Vcam-1 expression were measured. ROS production was increased and mitochondrial network was damaged without NF-κB, NO production or mitochondrial membrane potential alterations. Similar ultrastructural changes were seen in l-ornithine treated brain endothelial cells as in vivo. The basal negative zeta potential of brain endothelial cells became more positive after l-ornithine treatment.ConclusionWe demonstrated BBB damage in the l-ornithine-induced rat AP model suggesting a general, AP model independent effect. l-ornithine induced oxidative stress, decreased barrier integrity and altered BBB morphology in a culture BBB model. These data suggest a direct effect of the cationic l-ornithine on brain endothelium. Endothelial surface glycocalyx injury was revealed both in vivo and in vitro, as an additional novel component of the BBB-related pathological changes in AP.

Flow induces barrier and glycocalyx-related genes and negative surface charge in a lab-on-a-chip human blood-brain barrier model

Microfluidic lab-on-a-chip (LOC) devices allow the study of blood-brain barrier (BBB) properties in dynamic conditions. We studied a BBB model, consisting of human endothelial cells derived from hematopoietic stem cells in co-culture with brain pericytes, in an LOC device to study fluid flow in the regulation of endothelial, BBB and glycocalyx-related genes and surface charge. The highly negatively charged endothelial surface glycocalyx functions as mechano-sensor detecting shear forces generated by blood flow on the luminal side of brain endothelial cells and contributes to the physical barrier of the BBB. Despite the importance of glycocalyx in the regulation of BBB permeability in physiological conditions and in diseases, the underlying mechanisms remained unclear. The MACE-seq gene expression profiling analysis showed differentially expressed endothelial, BBB and glycocalyx core protein genes after fluid flow, as well as enriched pathways for the extracellular matrix molecules. We observed increased barrier properties, a higher intensity glycocalyx staining and a more negative surface charge of human brain-like endothelial cells (BLECs) in dynamic conditions. Our work is the first study to provide data on BBB properties and glycocalyx of BLECs in an LOC device under dynamic conditions and confirms the importance of fluid flow for BBB culture models.

Immediate effects of whole blood donation on the endothelial surface layer and glycocalyx shedding.

The endothelium plays a pivotal role in regulating microvascular function, especially in situations associated with acute blood loss. Whether blood donation and the associated volume loss affects the dimensions of the endothelial surface layer (ESL) and glycocalyx integrity remains unknown. This study was designed to determine real-time ESL and perfusion measurements of the sublingual microcirculation using sidestream dark field imaging performed in healthy subjects shortly before and after a donation of 500 mL whole blood. A novel image acquisition and analysis software (GlycoCheck™) automatically calculated the perfused boundary region (PBR), an inverse parameter for red blood cell (RBC) penetration into the ESL, in vessels between 5 and 25 μm in diameter. Microvascular perfusion was measured by RBC filling percentage. Soluble glycocalyx components were determined in the peripheral circulation. There was no significant immediate effect of whole blood donation on PBR and RBC filling percentage. Linear regression analysis revealed a distinct association between change in PBR and change in RBC filling percentage (regression coefficient β: -0.040; 95% confidence interval: -0.049 to -0.030; p&lt;0.001). A significant reduction in plasma heparan sulphate (1,329±316 vs 1,237±275 ng/mL, p=0.005) and hyaluronan (94±18 vs 90±16 ng/mL, p=0.002) was noted, while syndecan-1 levels (30 [23-50] vs 29 [24-46] ng/mL, p=0.282) remained unchanged. Dimensions and integrity of the ESL appear to remain stable following a 500 mL whole blood donation and reflect its ability to ensure microvascular function and perfusion.

SAT-368 Galactose-deficient IgA1 containing immune complexes deposit in mesangium via endothelial cell injuries

IgA nephropathy (IgAN) is the most common primary glomerulonephritis, and causes end-stage renal disease in 20–40% of patients within 20 years after onset of this disease. IgAN is defined by the presence of dominant or co-dominant mesangial IgA1 immune deposits, accompanied by C3 deposits, and deposition of IgA1 includes galactose-deficient IgA1 (Gd-IgA1). Serum levels of Gd-IgA1 and Gd-IgA1-containing immune complexes (IC) with Gd-IgA1-specific autoantibodies are elevated in patients with IgAN. However, the pathogenic role of Gd-IgA1-containing IC and mesangial immune deposits are still unclear. The endothelial surface glycocalyx modulates vascular function. Furthermore, loss of the glomerular endothelial glycocalyx links albuminuric kidney disease. Therefore, we examined that Gd-IgA1-containg ICs deposit in mesangium through glomerular endothelial cell disorder. Gd-IgA1 and recombinant anti-glycan IgG were used to form IC (Gd-IgA1-IgG IC) to inject i.v into nude mice. After various time intervals, mice were sacrificed and kidney was harvested to determine mesangial deposition and kidney injuries. Samples of urine were collected to determine urinary protein and hematuria. Furthermore, human renal glomerular endothelial cells (HRGEC) were co-cultured with Gd-IgA1 alone or Gd-IgA1-IgG IC for 72 h to assess the potential capacity of these IC to activate endothelial cells. Transcript levels of TNF-α, TGF-β, IL-6, ICAM1 and E-selectin in HRGEC were measured by Real-time PCR. Also, to investigate that Gd-IgA1-IgG IC stimulation increases permeability of glomerular microvascular resulted in renal injuries, the renal microvascular endothelial glycocalyx removal was estimated by real-time glycocalyx imaging. Gd-IgA1 alone or Gd-IgA1-IgG IC were injected into nude mice. The glycocalyx were observed for 45 min and after that mice were sacrificed to confirm glomerular deposition. Gd-IgA1 and anti-glycan IgG formed IC that deposited with murine C3 in the mesangium and in small amounts in the subendothelial area of the glomerular capillaries, and induced proteinuria and hematuria. Electron microscopic examination showed that injection of Gd-IgA1-IgG IC induced podocyte and endothelial injuries. Stimulation with Gd-IgA1-IgG IC upregulated transcript levels of TNF-α, IL-6, ICAM1 and E-selectin in HRGEC (P<0.01). Real-time glycocalyx imaging showed that Gd-IgA1-IgG IC removed renal microvascular glycocalyx immediately after injection. Furthermore, after stimulation with Gd-IgA1-IgG IC, IgA and C3 were deposited in mesangium, and proteinuria and hematuria were induced. Gd-IgA1-containg IC might bind to glomerular endothelial cells and induce release of pathogenic cytokines and chemokines. Furthermore, stimulation of Gd-IgA1-containg IC involved in endothelial dysfunction with complementary activation resulted in renal injuries.

Endothelial surface glycocalyx (ESG) components and ultra-structure revealed by stochastic optical reconstruction microscopy (STORM).

In order to play different roles in vascular functions as a mechanosensor to blood flows and as a barrier to transvascular exchange, the endothelial surface glycocalyx (ESG) should have an organized structure. Due to the limitations of optical and electron microscopy, the ultra-structure of ESG has not been revealed until the recent development of super-resolution optical microscopy, STORM. To investigate the ESG components and their organization on bEnd3 (mouse brain microvascular endothelial cells) monolayer. ESG was immunolabeled with anti-heparan sulfate (HS), followed by an ATTO488 conjugated goat anti-mouse IgG, and with biotinylated hyaluronic acid (HA) binding protein, followed by an AF647 conjugated anti-biotin. The ESG was then imaged by the STORM. HA is a long molecule weaving into a network which covers the endothelial luminal surface. In contrast, HS is a shorter molecule, perpendicular to the cell surface. HA and HS are partially overlapped with each other at the endothelial luminal surface. We also quantified the length, diameter, orientation, and density of HS at the top, middle and bottom regions of the endothelial surface. Our results suggest that HS plays a major role in mechanosensing and HA plays a major role in the molecular sieve.

The role of endothelial glycocalyx in sepsis

The surface of endothelial cells is filled with various membrane-bound molecules that form the glycocalyx. The endothelial glycocalyx is a surface layer mainly consisted of glycosaminoglycans that include heparan sulfate, chondroitin sulfate, and hyaluronic acid and its core proteins. Previous studies suggest that endothelial surface glycocalyx shedding could play a role in endothelial dysfunction and inflammation. This article will review the endothelial glycocalyx and its role in sepsis.

Pro-atherosclerotic disturbed flow disrupts caveolin-1 expression, localization, and function via glycocalyx degradation

BackgroundEndothelial-dependent atherosclerosis develops in a non-random pattern in regions of vessel bending and bifurcations, where blood flow exhibits disturbed flow (DF) patterns. In contrast, uniform flow (UF), normal endothelium, and healthy vessel walls co-exist within straight vessels. In clarifying how flow protectively or atherogenically regulates endothelial cell behavior, involvement of the endothelial surface glycocalyx has been suggested due to reduced expression in regions of atherosclerosis development. Here, we hypothesized that pro-atherosclerotic endothelial dysfunction occurs as a result of DF-induced reduction in glycocalyx expression and subsequently impairs endothelial sensitivity to flow. Specifically, we propose that glycocalyx degradation can induce pro-atherosclerotic endothelial dysfunction through decreased caveolin-1 and endothelial nitric oxide synthase expression and localization.MethodsWe studied endothelial cells in atherosclerotic-prone DF and atherosclerotic-resistant UF conditions in parallel plate flow culture and in C57Bl/6 mice. The effects of flow conditioning on endothelial cell behavior were quantified using immunocytochemistry. The glycocalyx was fluorescently labeled for wheat germ agglutinin, which serves as a general glycocalyx label, and heparan sulfate, a major glycocalyx component. Additionally, mechanosensitivity was assessed by immunocytochemical fluorescence expression and function of caveolin-1, the protein that forms the mechanosignaling caveolar invaginations on the endothelial surface, total endothelial-type nitric oxide synthase (eNOS), which synthesizes nitric oxide, and serine 1177 phosphorylated eNOS (eNOS-pS1177), which is the active form of eNOS. Caveolin function and eNOS expression and activation were correlated to glycocalyx expression. Heparinase III enzyme was used to degrade a major glycocalyx component, HS, to identify the role of the glycocalyx in caveoin-1 and eNOS-pS1177 regulation.ResultsResults confirmed that DF reduces caveolin-1 expression and abolishes most of its subcellular localization preferences, when compared to the effect of UF. DF down-regulates caveolin-1 mechanosignaling, as indicated by its reduced colocalization with serine 1177 phosphorylated endothelial-type nitric oxide synthase (eNOS-pS1177), a vasoregulatory signaling molecule whose activity is regulated by its residence in caveolae. As expected, DF inhibited glycocalyx expression compared to UF. In the absence of heparan sulfate, a major glycocalyx component, UF-conditioned endothelial cells exhibited near DF-like caveolin-1 expression, localization, and colocalization with eNOS-pS1177.ConclusionsThis is the first demonstration of a flow-defined role of the glycocalyx in caveolae expression and function related to vasculoprotective endothelial mechanosensitivity that defends against atherosclerosis. The results suggest that a glycocalyx-based therapeutic targeted to areas of atherosclerosis development could prevent disease initiation and progression.

Abstract 111: Endothelial Sodium Channel Activation Promotes Angiotensin II Induced Arterial Stiffness and Endothelial Dysfunction

Arterial stiffness is an independent risk factor for the development and progression of cardiovascular and kidney disease. Inappropriate activation of the renin-angiotensin-aldosterone system (RAAS) is seen in obesity and contributes significantly to development of arterial stiffness, vascular dysfunction and hypertension. We have recently observed that aldosterone increases the presence of epithelial sodium channels on endothelial cells (EnNaC), which results in stiffening of the endothelial cortical surface (glycocalyx) of the aorta. Accordingly, we posited that deletion of the critical alpha catalytic subunit of EnNaC would decrease Ang II induced arterial stiffening and vascular relaxation responses, in part, via decreases in oxidative stress. To investigate, we used mice with EC cell specific deletion of EnNaC, by serially crossing floxed” EnNaC mice with Tie 2-Cre transgene mice. EnNaC expression was markedly decreased in ECs from EnNaC KO mice while macrophage expression of EnNaC was unaffected. Ang II (500 ng/kg/min) was administered by infusion to 9 month old wild (WT) and EnNaC KO mice for three weeks with or without administration of the antioxidant tempol (2mM/day) in drinking water. Whole cell Na+ currents in ECs, as evaluated by patch clamp, were significantly increased by Ang II infusion, and this was markedly attenuated in EnNAC KO and tempol treated mice. Further, aortic endothelial stiffness as determined by atomic force microscopy was significantly increased in Ang II infused mice and this increase was attenuated in EnNaC KO mice. EnNaC deletion also attenuated impaired acetylcholine (eNOS)-mediated vasorelaxation. Thus, treatment with the antioxidant tempol reduced Ang II induced vascular stiffness and prevented impairment of NO mediated vascular relaxation. Moreover, tempol administration to Ang II infused EnNaC KO mice had similar effects in both Ang II-tempol and Ang II-EnNaC KO groups. Arterial stiffness was significantly decreased by EnNaC deletion or treatment with the antioxidant tempol in femoral vessels as well. These results are consistent with an important role for oxidative stress in Ang II promotion of EnNaC activation, endothelial stiffness and impaired eNOS mediated vascular relaxation.

Endothelial Surface Glycocalyx Revealed by Stochastic Optical Reconstruction Microscopy (STORM)

On the luminal surface of our blood vessels, there is a thin layer called endothelial surface glycocalyx (ESG) which consists of proteoglycans, glycosaminoglycans (GAGs) and glycoproteins. The GAGs in the ESG are heparan sulfate (HS), hyaluronic acid (HA), chondroitin sulfate (CS), and sialic acid (SA). In order to play important roles in vascular functions, such as being a mechanosensor and transducer for the endothelial cells (ECs) to sense the blood flow, a molecular sieve to maintain normal microvessel permeability and a barrier between the circulating cells and endothelial cells forming the vessel wall, the ESG should have an organized structure at the molecular level. Due to the limitations of optical and electrical microscopy, the ultra-structure of ESG has not been revealed until recent development of super high resolution fluorescence optical microscope, STORM. We used newly acquired Nikon-STORM system to observe the ESG on in vitro EC (bEnd3, mouse brain microvascular endothelial cells) monolayers. After confluent, the bEnd3 cells were immunolabeled with anti-HS, followed by an ATTO488 conjugated goat anti-mouse IgG, and with biotinylated HA binding protein, followed by an AF647 conjugated anti-biotin. The ESG was then imaged by the STORM with a 100 x/1.49 oil immersed lens. Multiple Reporters of ATTO488 and AF647 with alternating illumination were used to acquire the 3D images of HS and HA. The field of 256 × 256 (40 × 40 μm2) of HS and HA at the surface of ECs was obtained based on totally 40,000 of EM-CCD captured images for each reporter at a capturing speed of 19 ms/frame. The figure demonstrates the STORM images for the HA (top view, red, top row on the right) and HS (green, top row in the middle) and overlay (top row on the left) of the ESG focused at the bottom surface of the bEnd3 cells. The EC plasma membrane is located at the bottom plane of the 3D images (bottom row). The color bars in the 3D side views are the scale bars in nm. The figure shows that HA is a long molecule weaving into a network, which is horizontal to the EC cell surface. In contrast, HS is a shorter molecule, which is perpendicular to the cell surface. The height of the HS is ~600 nm. HA and HS seem to overlap with each other at the EC surface. The revealed ultra-structure of ESG by STORM suggests that HS plays a major role in mechanosensing and HA plays a major role in forming the molecular sieve. Support or Funding Information NIH 1SC1CA153325-01 and NSF MRI CBET 1337746 STORM images showing distribution of ATTO 488 labeled anti-HS (green, middle) and Alexa Fluor 647 labeled anti-HA (red, right) and overlay (left) in the ESG of bEnd3 cell monolayer, focused on the bottom surface of the cells. The EC plasma membrane is located at the bottom plane of the 3D images (bottom row). The color bars in the 3D side views are the scale bars in nm. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Role of Endothelial Surface Glycocalyx in Mechanosensing and Transduction.

The endothelial cells (ECs) forming the inner wall of every blood vessel are constantly exposed to the mechanical forces generated by blood flow. The EC responses to these hemodynamic forces play a critical role in the homeostasis of the circulatory system. A variety of mechanosensors and transducers, locating on the EC surface, intra- and trans-EC membrane, and within the EC cytoskeleton, have thus been identified to ensure proper functions of ECs. Among them, the most recent candidate is the endothelial surface glycocalyx (ESG), which is a matrix-like thin layer covering the luminal surface of the EC. It consists of various proteoglycans, glycosaminoglycans, and plasma proteins and is close to other prominent EC mechanosensors and transducers. This chapter summarizes the ESG composition, thickness, and structure observed by different labeling and visualization techniques and in different types of vessels. It also presents the literature in determining the ESG mechanical properties by atomic force microscopy and optical tweezers. The molecular mechanisms by which the ESG plays the role in EC mechanosensing and transduction are described as well as the ESG remodeling by shear stress, the actin cytoskeleton, the membrane rafts, the angiogenic factors, and the sphingosine-1-phosphate.

Sphingosine-1-phosphate reduces adhesion of malignant mammary tumor cells MDA-MB-231 to microvessel walls by protecting endothelial surface glycocalyx.

Sphingosine-1-phosphate (S1P) is a sphingolipid in plasma that plays a critical role in cardiovascular and immune systems. Endothelial surface glycocalyx (ESG) decorating the inner wall of blood vessels is a regulator of multiple vascular functions. To test the hypothesis that S1P can reduce tumor cell adhesion to microvessel walls by protecting the ESG, we quantified the ESG and MDA-MB-231 tumor cell adhesion in the presence and absence of 1μM S1P, and in the presence of the matrix metalloproteinase (MMP) inhibitor in post-capillary venules of rat mesentery. We also measured the microvessel permeability to albumin as an indicator for the microvessel wall integrity. In the absence of S1P, ESG was ~10% of that in the presence of S1P, whereas adherent tumor cells and the permeability to albumin and were ~3.5-fold (after 30 min adhesion) and ~7.7-fold that in the presence of S1P, respectively. In the presence of the MMP inhibitor, the results are similar to those in the presence of S1P. Our results conform to the hypothesis that protecting ESG by S1P inhibits MDA-MB-231 tumor cell adhesion to the microvessel wall.

Sphingosine‐1‐phosphate reduces adhesion of malignant mammary tumor cells MDA‐MB‐231 to microvessel walls by protecting endothelial surface glycocalyx

To test the hypothesis that the plasma phospholipid S1P can reduce tumor cell adhesion to microvessel walls by protecting the endothelial surface glycocalyx (ESG), we quantified the ESG by fluorescence immunostaining of heparan sulfate (HS), the most abundant glycosaminoglycans in the ESG, the microvessel permeability to albumin, and MDA‐MB‐231 tumor cell adhesion in the presence and absence of S1P in post‐capillary venules of rat mesentery. Rats (SD, 250–300g) were anesthetized with pentobarbital sodium given subcutaneously and kept warm on a heating pad. A midline incision (~2 inch) was made in the abdominal wall and the mesentery was gently taken out from the abdominal cavity and arranged on the surface of a glass coverslip for the measurement. A post‐capillary venule of 35–50μm diameter was cannulated by a glass micropipette; after 20 min perfusing with 1% fatty acid free bovine serum albumin Ringer solution with and without 1 μM S1P, the intensity of FITC‐anti‐HS, permeability to albumin and tumor cell adhesion rates were quantified, respectively, in 3 groups of experiments. In the absence of S1P, FITC‐anti‐HS labeled ESG was ~10% of that in the presence of S1P, whereas the permeability to albumin and adherent tumor cells were ~7‐fold and ~3.5‐fold (after 30 min adhesion) those in the presence of S1P, respectively. Our results conform to the hypothesis that protecting ESG by S1P inhibits tumor cell adhesion to the microvessel wall.Support or Funding InformationSupported by NIH SC1CA153325.

Mechanotransduction of the endothelial glycocalyx mediates nitric oxide production through activation of TRP channels.

The endothelial surface glycocalyx (ESG) is a carbohydrate-rich layer found on the vascular endothelium, serving critical functions in the mechanotransduction of blood flow-induced forces. One of the most important protective functions of the ESG is to mediate the production of nitric oxide (NO) in response to blood flow. However, the detailed mechanism underlying ESG's mechanotransduction of the production of NO has not been completely identified. Herein, using the cultured rat brain microvascular endothelial cells (bEnd.3) as a model system, we have implemented a combined atomic force and fluorescence microscopy approach to show that the ESG senses and transduces vertical mechanical stretch to produce NO. This rapid NO production is dependent on the presence of both heparan sulfate (HS) and hyaluronic acid (HA) in ESG, as the removal of HS and/or HA leads to a significant decrease in NO production. Moreover, the production of NO is dependent on the intake of Ca2+ via endothelial transient receptor potential (TRP) channels. Together, our results demonstrate the molecular mechanism of rapid production of NO in response to vertical mechanical stretch.

Dual Regulation of L-Selectin-Mediated Leukocyte Adhesion by Endothelial Surface Glycocalyx.

Endothelial surface glycocalyx (ESG) is a carbohydrate-rich, gel-like layer found on vascular endothelium, serving critical functions in mechanotransduction of blood flows, maintenance of the endothelial permeability, and the control of leukocyte adhesion and inflammation. This study aimed to clarify the role of ESG in the adhesion between leukocytes and Human Umbilical Vein Endothelial Cells (HUVECs) under resting or inflammatory conditions. Using an atomic force microscopy-based single-cell adhesion assay, we directly quantified the detachment force and work perpendicular to the cell membrane. Detachment force and work were measured for every separation event of a leukocyte from a HUVEC with ESG, or with the major ESG glycosaminoglycan components, heparan sulfate (HS) and hyaluronic acid (HA) removed. For the resting HUVECs, when HS and/or HA were removed, the detachment force and work increased dramatically. For the HUVECs activated by inflammatory cytokine tumor necrosis factor alpha, we observed increases in the detachment force and work compared to the resting HUVECs, and removal of HS and/or HA resulted in significant decreases in the detachment force and work. The results demonstrate that the ESG layer serves a dual function: (1) on resting endothelium, it prevents leukocyte adhesion, and (2) under inflammatory conditions, it participates in endothelial-leukocyte interactions with molecules such as selectins.

Endothelial glycocalyx, apoptosis and inflammation in an atherosclerotic mouse model

Background and aimsPrevious experiments suggest that both increased endothelial cell apoptosis and endothelial surface glycocalyx shedding could play a role in the endothelial dysfunction and inflammation of athero-prone regions of the vasculature. We sought to elucidate the possibly synergistic mechanisms by which endothelial cell apoptosis and glycocalyx shedding promote atherogenesis. Methods4- to 6-week old male C57Bl/6 apolipoprotein E knockout (ApoE−/−) mice were fed a Western diet for 10 weeks and developed plaques in their brachiocephalic arteries. ResultsGlycocalyx coverage and thickness were significantly reduced over the plaque region compared to the non-plaque region (coverage plaque: 71 ± 23%, non-plaque: 97 ± 3%, p = 0.02; thickness plaque: 0.85 ± 0.15 μm, non-plaque: 1.2 ± 0.21 μm, p = 0.006). Values in the non-plaque region were not different from those found in wild type mice fed a normal diet (coverage WT: 92 ± 3%, p = 0.7 vs. non-plaque ApoE−/−, thickness WT: 1.1 ± 0.06 μm, p = 0.2 vs. non-plaque ApoE−/−). Endothelial cell apoptosis was significantly increased in ApoE−/− mice compared to wild type mice (ApoE−/−:64.3 ± 33.0, WT: 1.1 ± 0.5 TUNEL-pos/cm, p = 2 × 10−7). The number of apoptotic endothelial cells per unit length was 2 times higher in the plaque region than in the non-plaque region of the same vessel (p = 3 × 10−5). Increased expression of matrix metalloproteinase 9 co-localized with glycocalyx shedding and plaque buildup. ConclusionsOur results suggest that, in concert with endothelial apoptosis that increases lipid permeability, glycocalyx shedding initiated by inflammation facilitates monocyte adhesion and macrophage infiltration that promote lipid retention and the development of atherosclerotic plaques.

LBM simulations on the influence of endothelial SGL structure on cell adhesion in the micro-vessels

The endothelial surface glycocalyx layer (SGL) plays a crucial role in modulating vascular permeability, sensing hydrodynamic changes and attenuating cell adhesion in microcirculation. In this work, the effect of endothelial SGL structure on cell adhesion was numerically studied in the uneven micro-vessel caused by the shedding of SGL. The blood dynamics was conducted by the lattice Boltzmann method (LBM), the adhesive dynamics was performed by the stochastic Monte Carlo method, and the cell dynamics was implemented by the Newton′s law with translation and rotation. To validate the numerical schemes, the cell suspension flow in the symmetric stenotic vessel and the leukocyte adhesion in the straight micro-vessel were executed firstly. The simulation results show that the present numerical schemes are capable of studying cell suspension behavior and leukocyte adhesive phenomenon in the 2-D blood vessels. By investigating the effect of SGL structure on cell adhesion, it is found that the endothelial SGL structure significantly influences cell adhesion function, by changing the geometry of the vessel, providing more ligands to form receptor–ligand bonds, and enhancing the forward reaction rate for forming new bonds. The present results may be helpful in understanding the mechanical mechanism of cell adhesion function with the shedding of endothelial SGL in microcirculation.