scholarly journals Changes in endothelial glycocalyx layer protective ability after inflammatory stimulus

2020 ◽  
Author(s):  
Luis F. Delgadillo ◽  
Elena B. Lomakina ◽  
Julia Kuebel ◽  
Richard E. Waugh
Keyword(s):  
Endothelial Cells ◽  
Fluid Transport ◽  
Leukocyte Adhesion ◽  
Umbilical Vein ◽  
Barrier Properties ◽  
Endothelial Barrier ◽  
Endothelial Glycocalyx ◽  
Neutrophil Adhesion ◽  
Barrier Dysfunction ◽  
Endothelial Glycocalyx Layer

Leukocyte adhesion to the endothelium is an important early step in the initiation and progression of sepsis. The endothelial glycocalyx layer (EGL) has been implicated in neutrophil adhesion and barrier dysfunction, but studies in this area are few. In this report we examine the hypothesis that damage to the structure of the EGL caused by inflammation leads to increased leukocyte adhesion and endothelial barrier dysfunction. We used human umbilical vein endothelial cells (HUVECs) enzymatically treated to remove the EGL components hyaluronic acid (HA) and heparan sulfate (HS) as a model for EGL damage. Using atomic force microscopy, we show reductions in EGL thickness after removal of either HA or HS individually, but the largest decrease, comparable to TNF-a treatment, was observed when both HA and HS were removed. Interestingly, removal of HS or HA individually did not affect neutrophil adhesion significantly, but removal of both constituents resulted in increased neutrophil adhesion. To test EGL contributions to endothelial barrier properties, we measured trans-endothelial electrical resistance (TEER) and diffusion of fluorescently labeled dextran (10 kDa MW) across the monolayer. Removal of EGL components decreased TEER, but had an insignificant effect on dextran diffusion rates. The reduction in TEER suggests that disruption of the EGL may predispose endothelial cells to increased rates of fluid leakage. These data support the view that damage to the EGL during inflammation has significant effects on the accessibility of adhesion molecules, likely facilitates leukocyte adhesion, and may also contribute to increased rates of fluid transport into tissues.

Fluid shear stress stimulates incorporation of hyaluronan into endothelial cell glycocalyx

2006 ◽  
Vol 290 (1) ◽  
pp. H458-H452 ◽  
Author(s):  
Mirella Gouverneur ◽  
Jos A. E. Spaan ◽  
Hans Pannekoek ◽  
Ruud D. Fontijn ◽  
Hans Vink
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Fluid Shear Stress ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Barrier Properties ◽  
Endothelial Glycocalyx ◽  
Stable Cell Line ◽  
Permeability Barrier ◽  
Fluid Shear

Vascular endothelial cells are shielded from direct exposure to flowing blood by the endothelial glycocalyx, a highly hydrated mesh of glycoproteins, sulfated proteoglycans, and associated glycosaminoglycans (GAGs). Recent data indicate that the incorporation of the unsulfated GAG hyaluronan into the endothelial glycocalyx is essential to maintain its permeability barrier properties, and we hypothesized that fluid shear stress is an important stimulus for endothelial hyaluronan synthesis. To evaluate the effect of shear stress on glycocalyx synthesis and the shedding of its GAGs into the supernatant, cultured human umbilical vein endothelial cells (i.e., the stable cell line EC-RF24) were exposed to 10 dyn/cm2 nonpulsatile shear stress for 24 h, and the incorporation of [3H]glucosamine and Na2[35S]O4 into GAGs was determined. Furthermore, the amount of hyaluronan in the glycocalyx and in the supernatant was determined by ELISA. Shear stress did not affect the incorporation of 35S but significantly increased the amount of glucosamine-containing GAGs incorporated in the endothelial glycocalyx [168 (SD 17)% of static levels, P < 0.01] and shedded into the supernatant [231 (SD 41)% of static levels, P < 0.01]. Correspondingly with this finding, shear stress increased the amount of hyaluronan in the glycocalyx [from 26 (SD 24) × 10−4 to 46 (SD 29) × 10−4 ng/cell, static vs. shear stress, P < 0.05] and in the supernatant [from 28 (SD 11) × 10−4 to 55 (SD 16) × 10−4 ng·cell−1·h−1, static vs. shear stress, P < 0.05]. The increase in the amount of hyaluronan incorporated in the glycocalyx was confirmed by a threefold higher level of hyaluronan binding protein within the glycocalyx of shear stress-stimulated endothelial cells. In conclusion, fluid shear stress stimulates incorporation of hyaluronan in the glycocalyx, which may contribute to its vasculoprotective effects against proinflammatory and pro-atherosclerotic stimuli.

scholarly journals Platelet-dependent primary hemostasis promotes selectin- and integrin- mediated neutrophil adhesion to damaged endothelium under flow conditions

Blood ◽  
1996 ◽  
Vol 87 (8) ◽  
pp. 3271-3281 ◽  
Author(s):  
PH Kuijper ◽  
HI Gallardo Torres ◽  
JA van der Linden ◽  
JW Lammers ◽  
JJ Sixma ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Vessel Wall ◽  
Platelet Adhesion ◽  
Leukocyte Adhesion ◽  
Umbilical Vein ◽  
Blood Platelets ◽  
Neutrophil Adhesion ◽  
Shear Stresses ◽  
Flow Conditions

Co-localization of blood platelets and granulocytes at sites of hemostasis and inflammation has triggered an intense interest in possible interactions between these cellular processes and induction of vessel wall injury. Leukocyte adhesion to endothelial cells decreases with increasing shear and is dependent on an initial rolling phase mediated by selectins. We hypothesized that flow-dependent platelet adhesion at an injured vessel wall will lead to P-selectin expression by platelets, thus mediating leukocyte co-localization. A perfusion chamber was used in which flowing whole blood induced platelet adhesion to a subendothelial matrix (ECM) of cultured human umbilical vein endothelial cells (HUVEC). We compared neutrophil (polymorphonuclear leukocyte [PMN]) interactions with HUVEC and their ECM with and without adhered platelets. PMNs adhered predominantly to ECM-adhered platelets and not to endothelial cells. ECM alone did not support PMN adhesion under flow conditions. PMN adhesion to unstimulated HUVEC was only substantial at low shear (up to 200 cells/mm2 at shear stress 80 mPa). In marked contrast, PMN adhesion to ECM-adhered platelets was dramatically increased, and adhesion was demonstrated at much higher shear stress (up to 640 mPa). Studies with specific antibodies showed that the platelet-dependent neutrophil adhesion was selectin-mediated. Inhibition of P-selectin caused a marked inhibition of adhesion at high shear stress, whereas the role of leukocyte L-selectin was less pronounced. beta2-Integrin-blocking antibodies inhibited static neutrophil adhesion. fMLP induced L-selectin shedding from leukocytes, resulting in decreased leukocyte adhesion. In conclusion, platelet- dependent hemostasis at the ECM appears to be a powerful intermediate in neutrophil-vessel wall interactions at shear stresses that normally do not allow neutrophil adhesion to intact endothelium.

Sickle Red Blood Cell Induced Adhesion of Neutrophils to Endothelial Cells and Biologic Correlates of Leukocyte Activation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1055-1055 ◽  
Author(s):  
Lydia A. Boateng ◽  
Rahima Zennadi ◽  
Marilyn J. Telen
Keyword(s):  
Endothelial Cells ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Leukocyte Count ◽  
Leukocyte Adhesion ◽  
Umbilical Vein ◽  
Direct Interaction ◽  
Total Leukocyte Count ◽  
Neutrophil Adhesion ◽  
Leukocyte Activation

Abstract 1055 In sickle cell disease (SCD), any event that slows the passage of red blood cells (RBCs) in the microcirculation can promote hemoglobin polymerization, red cell sickling, and vaso-occlusion. Homozygous (SS) RBCs have a sticky surface and, compared to normal (AA) RBCs, attach readily to both endothelial cells and other blood cells. Furthermore, the adhesive function of SS RBCs is up-regulated by the stress hormone epinephrine, resulting in significant vaso-occlusion and increased mononuclear leukocyte adhesion to the vascular endothelium in a mouse model. We therefore postulated that direct interaction between SS RBCs and neutrophils may also lead to activation and adhesion of neutrophils to endothelial cells. In order to explore whether SS RBCs can induce adhesion of neutrophils to endothelial cells independent of exogenous cytokine stimulation, neutrophils were isolated from healthy donors using density gradient media and then labeled with fluorescent dye. Fluorescent neutrophils were then co-incubated with epinephrine-treated SS or sham-treated (untreated) SS, AA or no added RBCs (control) at 37°C. Graduated height flow chambers were used to quantify adhesion of neutrophils to human umbilical vein endothelial cells (HUVECs) grown on glass slides coated with gelatin. We then also examined markers of hematologic status and endothelial activation to determine if any of these were correlated with the ability of SS RBCs to induce neutrophil adhesion. Enzyme linked immunosorbent assays (ELISAs) were used to quantify inflammatory markers (IL-1B, IL-6, IL-8), soluble L selectin (sL-SEL) and soluble P selectin (sP-SEL) in the plasma of the SS patient samples used in adhesion assays. We found that neither sham-treated nor epi-treated AA RBCs (n=3) significantly increased neutrophil adhesion to HUVECs, while both sham-treated and epinephrine-stimulated (epi) SS RBCs significantly increased neutrophil adhesion to endothelial cells (n=17, 42.0% adhesion for epi-SS RBCs vs 20.0% for no RBCs and 27.2% for untreated SS RBCs, with p values as shown in Figure 1). ELISA immunoassays confirmed elevated levels of sP-SEL and sL-SEL in SS patients, and levels of sL-SEL correlated with total leukocyte count (p=0.005). However, neither these biomarkers nor leukocyte count correlated with the ability of patient RBCs to stimulate normal leukocyte adhesion. Levels of IL-1B, IL-6 and IL-8 also did not correlate with the ability of patients' RBCs to stimulate neutrophil adhesion. However, we also found that HbF levels correlated inversely with sL-SEL levels (P=0.014), suggesting that HbF not only helps prevent RBC sickling but also reduces leukocyte activation. Other hematologic markers (Hb level, platelet count, reticulocyte count, LDH) also did not correlate with the ability of epi-SS RBCs to induce neutrophil adhesion. Therefore, we conclude that SS RBCs are able to stimulate neutrophil adhesion, especially under conditions that activate the adhesion receptors responsible for RBC-neutrophil interaction. In addition, we found that sL-SEL was increased in the presence of higher WBC counts, while it was inversely related to HbF levels. Taken together, these data suggest that the effect of HbF on SS RBCs reduces leukocyte activation, and this may occur in part through reduction of SS RBC-leukocyte interaction. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Pigment Epithelium-Derived Factor Induces Endothelial Barrier Dysfunction via p38/MAPK Phosphorylation

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Ting He ◽  
Liping Zhao ◽  
Dongxia Zhang ◽  
Qiong Zhang ◽  
Jiezhi Jia ◽  
...  
Keyword(s):  
P38 Mapk ◽  
Umbilical Vein ◽  
Pigment Epithelium ◽  
Endothelial Barrier ◽  
Dependent Manner ◽  
Barrier Dysfunction ◽  
Edema Formation ◽  
Endothelial Barrier Dysfunction ◽  
Pigment Epithelium Derived Factor

Endothelial barrier dysfunction, which is a serious problem that occurs in various inflammatory conditions, permits extravasation of serum components into the surrounding tissues, leading to edema formation and organ failure. Pigment epithelium-derived factor (PEDF), which is a major endogenous antagonist, has been implicated in diverse biological process, but its role in endothelial barrier dysfunction has not been defined. To assess the role of PEDF in the vasculature, we evaluated the effects of exogenous PEDF using human umbilical vein endothelial cells (HUVECs)in vitro. Our results demonstrated that exogenous PEDF activated p38/MAPK signalling pathway in a dose- and time-dependent manner and induced vascular hyperpermeability as measured by the markedly increased FITC-dextran leakage and the decreased transendothelial electrical resistance (TER) across the monolayer cells, which was accompanied by microtubules (MTs) disassembly and F-actin rearrangement. However, the aforementioned alterations can be arrested by the application of low concentration of p38/MAPK inhibitor SB203580. These results reveal a novel role for PEDF as a potential vasoactive substance in inducing hyperpermeability. Furthermore, our results suggest that PEDF and p38/MAPK may serve as therapeutic targets for maintaining vascular integrity.

N-methyl-d-aspartate receptor activation in human cerebral endothelium promotes intracellular oxidant stress

2005 ◽  
Vol 288 (4) ◽  
pp. H1893-H1899 ◽  
Author(s):  
Christopher D. Sharp ◽  
J. Houghton ◽  
J. W. Elrod ◽  
A. Warren ◽  
T. H. Jackson ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Nmda Receptor ◽  
Oxidant Stress ◽  
Receptor Activation ◽  
Endothelial Barrier ◽  
Barrier Dysfunction ◽  
Membrane Pore ◽  
Cerebral Endothelial Cells ◽  
Ros Formation ◽  
Cytochrome P 450

Cerebral endothelial cells in the rat, pig, and, most recently, human have been shown to express several types of receptors specific for glutamate. High levels of glutamate disrupt the cerebral endothelial barrier via activation of N-methyl-d-aspartate (NMDA) receptors. We have previously suggested that this glutamate-induced barrier dysfunction was oxidant dependent. Here, we provide evidence that human cerebral endothelial cells respond to glutamate by generating an intracellular oxidant stress via NMDA receptor activation. Cerebral endothelial cells loaded with the oxidant-sensitive probe dihydrorhodamine were used to measure intracellular reactive oxygen species (ROS) formation in response to glutamate receptor agonists, antagonists, and second message blockers. Glutamate (1 mM) significantly increased ROS formation compared with sham controls (30 min). This ROS response was significantly reduced by 1) MK-801, a noncompetitive NMDA receptor antagonist; 2) 8-( N, N-diethylamino)- n-octyl-3,4,5-trimethoxybenzoate, an intracellular Ca2+ antagonist; 3) LaCl3, an extracellular Ca2+ channel blocker; 4) diphenyleiodonium, a heme-ferryl-containing protein inhibitor; 5) itraconazole, a cytochrome P-450 3A4 inhibitor; and 6) cyclosporine A, which prevents mitochondrial membrane pore transition required for mitochondrial-dependent ROS generation. Our results suggest that the cerebral endothelial barrier dysfunction seen in response to glutamate is Ca2+ dependent and may require several intracellular signaling events mediated by oxidants derived from reduced nicotinamide adenine dinucleotide oxidase, cytochrome P-450, and the mitochondria.

Analysis of tight junctions during neutrophil transendothelial migration

2000 ◽  
Vol 113 (1) ◽  
pp. 45-57 ◽  
Author(s):  
A.R. Burns ◽  
R.A. Bowden ◽  
S.D. MacDonell ◽  
D.C. Walker ◽  
T.O. Odebunmi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tight Junctions ◽  
Umbilical Vein ◽  
Interleukin 1 ◽  
Intercellular Junctions ◽  
Barrier Properties ◽  
Transendothelial Migration ◽  
Immunoblot Analysis ◽  
Intimate Contact ◽  
And Migration

Intercellular junctions have long been considered the main sites through which adherent neutrophils (PMNs) penetrate the endothelium. Tight junctions (TJs; zonula occludens) are the most apical component of the intercellular cleft and they form circumferential belt-like regions of intimate contact between adjacent endothelial cells. Whether PMN transmigration involves disruption of the TJ complex is unknown. We report here that endothelial TJs appear to remain intact during PMN adhesion and transmigration. Human umbilical vein endothelial cell (HUVEC) monolayers, a commonly used model for studying leukocyte trafficking, were cultured in astrocyte-conditioned medium to enhance TJ expression. Immunofluorescence microscopy and immunoblot analysis showed that activated PMN adhesion to resting monolayers or PMN migration across interleukin-1-treated monolayers does not result in widespread proteolytic loss of TJ proteins (ZO-1, ZO-2, and occludin) from endothelial borders. Ultrastructurally, TJs appear intact during and immediately following PMN transendothelial migration. Similarly, transendothelial electrical resistance is unaffected by PMN adhesion and migration. Previously, we showed that TJs are inherently discontinuous at tricellular corners where the borders of three endothelial cells meet and PMNs migrate preferentially at tricellular corners. Collectively, these results suggest that PMN migration at tricellular corners preserves the barrier properties of the endothelium and does not involve widespread disruption of endothelial TJs.

scholarly journals hnRNPA2/B1 Ameliorates LPS-Induced Endothelial Injury through NF-κB Pathway and VE-Cadherin/β-Catenin Signaling Modulation In Vitro

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Yi Chen ◽  
Dan Tang ◽  
Linjie Zhu ◽  
Tianjie Yuan ◽  
Yingfu Jiao ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Inflammatory Responses ◽  
Umbilical Vein ◽  
Cell Metabolism ◽  
Endothelial Permeability ◽  
Underlying Mechanism ◽  
Endothelial Injury ◽  
Inflammatory Factors ◽  
Barrier Dysfunction

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) is a protein involved in the regulation of RNA processing, cell metabolism, migration, proliferation, and apoptosis. However, the effect of hnRNPA2/B1 on injured endothelial cells (ECs) remains unclear. We investigated the effect of hnRNPA2/B1 on lipopolysaccharide- (LPS-) induced vascular endothelial injury in human umbilical vein endothelial cells (HUVECs) and the underlying mechanisms. LPS was used to induce EC injury, and the roles of hnRNPA2/B1 in EC barrier dysfunction and inflammatory responses were measured by testing endothelial permeability and the expression of inflammatory factors after the suppression and overexpression of hnRNPA2/B1. To explore the underlying mechanism by which hnRNPA2/B1 regulates endothelial injury, we studied the VE-cadherin/β-catenin pathway and NF-κB activation in HUVECs. The results showed that hnRNPA2/B1 was elevated in LPS-stimulated HUVECs. Moreover, knockdown of hnRNPA2/B1 aggravated endothelial injury by increasing EC permeability and promoting the secretion of the inflammatory cytokines TNF-α, IL-1β, and IL-6. Overexpression of hnRNPA2/B1 can reduce the permeability and inflammatory response of HUVEC stimulated by LPS in vitro, while increasing the expression of VE-Cadherin and β-catenin. Furthermore, the suppression of hnRNPA2/B1 increased the LPS-induced NF-κB activation and reduced the VE-cadherin/β-catenin pathway. Taken together, these results suggest that hnRNPA2/B1 can regulate LPS-induced EC damage through regulating the NF-κB and VE-cadherin/β-catenin pathways.

scholarly journals FBXW7 regulates endothelial barrier function by suppression of the cholesterol synthesis pathway and prenylation of RhoB

2019 ◽  
Vol 30 (5) ◽  
pp. 607-621 ◽  
Author(s):  
Manon C. A. Pronk ◽  
Jisca Majolée ◽  
Anke Loregger ◽  
Jan S. M. van Bezu ◽  
Noam Zelcer ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Barrier Function ◽  
Rho Gtpases ◽  
Umbilical Vein ◽  
Cholesterol Biosynthesis ◽  
Endothelial Barrier ◽  
Biosynthesis Pathway ◽  
Endothelial Barrier Function ◽  
Cholesterol Biosynthesis Pathway

Rho GTPases control both the actin cytoskeleton and adherens junction stability and are recognized as essential regulators of endothelial barrier function. They act as molecular switches and are primarily regulated by the exchange of GDP and GTP. However, posttranslational modifications such as phosphorylation, prenylation, and ubiquitination can additionally alter their localization, stability, and activity. F-box proteins are involved in the recognition of substrate proteins predestined for ubiquitination and subsequent degradation. Given the importance of ubiquitination, we studied the effect of the loss of 62 members of the F-box protein family on endothelial barrier function in human umbilical vein endothelial cells. Endothelial barrier function was quantified by electrical cell impedance sensing and macromolecule passage assay. Our RNA interference–based screen identified FBXW7 as a key regulator of endothelial barrier function. Mechanistically, loss of FBXW7 induced the accumulation of the RhoB GTPase in endothelial cells, resulting in their increased contractility and permeability. FBXW7 knockdown induced activation of the cholesterol biosynthesis pathway and changed the prenylation of RhoB. This effect was reversed by farnesyl transferase inhibitors and by the addition of geranylgeranyl pyrophosphate. In summary, this study identifies FBXW7 as a novel regulator of endothelial barrier function in vitro. Loss of FBXW7 indirectly modulates RhoB activity via alteration of the cholesterol biosynthesis pathway and, consequently, of the prenylation status and activity of RhoB, resulting in increased contractility and disruption of the endothelial barrier.

scholarly journals Soluble CD14 participates in the response of cells to lipopolysaccharide.

1992 ◽  
Vol 176 (6) ◽  
pp. 1665-1671 ◽  
Author(s):  
E A Frey ◽  
D S Miller ◽  
T G Jahr ◽  
A Sundan ◽  
V Bazil ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Leukocyte Adhesion ◽  
Umbilical Vein ◽  
Cell Types ◽  
Mononuclear Phagocytes ◽  
Soluble Cd14 ◽  
Bovine Endothelial Cells ◽  
Endothelial Leukocyte Adhesion Molecule ◽  
Umbilical Vein Endothelial Cells ◽  
Serum Dependent

CD14 is a 55-kD protein found both as a glycosylphosphatidyl inositol-linked protein on the surface of mononuclear phagocytes and as a soluble protein in the blood. CD14 on the cell membrane (mCD14) has been shown to serve as a receptor for complexes of lipopolysaccharide (LPS) with LPS binding protein, but a function for soluble CD14 (sCD14) has not been described. Here we show that sCD14 enables responses to LPS by cells that do not express CD14. We have examined induction of endothelial-leukocyte adhesion molecule 1 expression by human umbilical vein endothelial cells, interleukin 6 secretion by U373 astrocytoma cells, and cytotoxicity of bovine endothelial cells. None of these cell types express mCD14, yet all respond to LPS in a serum-dependent fashion, and all responses are completely blocked by anti-CD14 antibodies. Immunodepletion of sCD14 from serum prevents responses to LPS, and the responses are restored by addition of sCD14. These studies suggest that a surface anchor is not needed for the function of CD14 and further imply that sCD14 must bind to additional proteins on the cell surface to associate with the cell and transduce a signal. They also indicate that sCD14 may have an important role in potentiating responses to LPS in cells lacking mCD14.

Sign in / Sign up

Export Citation Format

Share Document