Validity of a patient-derived system of tissue-specific human endothelial cells: interleukin-6 as a surrogate marker in the coronary system

The aim of our study was to evaluate the relevance of tissue- and species-specific endothelial cells (EC) to study EC-dependent mechanisms in inflammatory-mediated tissue injury. We established an isolation protocol for highly purified EC (pEC) preparations of different origin and compared EC-specific inflammatory responses. Fluorescence-activated cell separation was used to obtain pEC cultures from different human arterial (coronary artery, internal thoracic artery) and venous (umbilical vein, saphenous vein) vessels. All pEC were analyzed for growth kinetics, morphology, release of cytokines/chemokines, and expression of E-selectin. For all different EC cultures, purities of ≥99% were reproducibly achieved. The EC isolation did not affect EC growth, morphology, and function. However, characterization of pEC from different vessel materials revealed an intrinsic, tissue-specific functional heterogeneity of EC cultures. Despite an arterial and venous difference in the secretion of IL-8 and monocyte chemoattractant protein-1, especially EC from coronary arteries produced significantly more IL-6 compared with other EC types, independent of age, gender, and disease of the cell donors. In contrast, the expression of E-selectin was not affected. We conclude that the proposed isolation protocol allows the generation of a pEC bank, enabling us to study tissue-specific aspects at the level of the endothelium.

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Assembly and Activation of the Intrinsic Fibrinolytic Pathway on the Surface of Human Endothelial Cells in Culture

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649800 ◽

1995 ◽

Vol 74 (02) ◽

pp. 698-703 ◽

Cited By ~ 29

Author(s):

Catherine Lenich ◽

Ralph Pannell ◽

Victor Gurewich

Keyword(s):

Endothelial Cells ◽

Umbilical Vein ◽

Factor Xii ◽

Plasminogen Activation ◽

High Molecular Weight Kininogen ◽

Human Endothelial Cells ◽

Intrinsic Pathway ◽

Local Fibrinolysis ◽

Umbilical Vein Endothelial Cells ◽

And Function

SummaryFactor XII has long been implicated in the intrinsic pathway of fibrinolysis, but the mechanism by which it triggers plasminogen activation and targets fibrinolysis has not been established. In the present study, the assembly and function of activated Factor XII (F.XIIa), prourokinase (pro-u-PA), high molecular weight kininogen (H-kininogen), and prekallikrein on human umbilical vein endothelial cells (HUVEC) was investigated. 125I-prekallikrein was shown to bind to HUVEC via receptor-bound H-kininogen in the presence of 50 μM ZnCl2. After the addition of F.XIIa, 78% of the 125I-prekallikrein initially bound to HUVEC was converted to 125I-kallikrein. However, only 6% of the HUVEC-bound 125I-pro-u-PA was thereby activated. This discrepancy was shown to be related to rapid dissociation (>50% within 15 min) of prekallikrein/kallikrein, but not pro-u-PA, from HUVEC. Increasing the level of cell-bound kallikrein increased the portion of cell-bound pro-u-PA activated, indicating that their co-localization was important for this pathway. Finally, F.XIIa was shown to trigger plasminogen activation on HUVEC via this pathway. This assembly of reactants on the endothelium suggests a mechanism whereby local fibrinolysis may be triggered by blood coagulation.

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Partial Characterization of a Fibrinolytic Inhibitor Produced by Cultured Endothelial Cells Derived from Human Umbilical Vein

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657146 ◽

1982 ◽

Vol 47 (02) ◽

pp. 128-131 ◽

Cited By ~ 14

Author(s):

F Esnard ◽

E Dupuy ◽

A M Dosne ◽

E Bodevin

Keyword(s):

Endothelial Cells ◽

Primary Culture ◽

Ammonium Sulphate ◽

Concanavalin A ◽

Umbilical Vein ◽

Human Umbilical Vein ◽

Preliminary Characterization ◽

Umbilical Vein Endothelial Cells ◽

Ammonium Sulphate Saturation

SummaryA preliminary characterization of a fibrinolytic inhibitor released by human umbilical vein endothelial cells in primary culture is reported. This molecule of Mr comprised between 2 × 105 and 106 and of μ2 mobility precipitates at 43% ammonium sulphate saturation and is totally adsorbed on Concanavalin A Sepharose 4 B. A possible relationship with a macroglobulins is discussed.

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Molecular and phenotypic analysis of rodent models reveals conserved and species-specific modulators of human sarcopenia

Communications Biology ◽

10.1038/s42003-021-01723-z ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Anastasiya Börsch ◽

Daniel J. Ham ◽

Nitish Mittal ◽

Lionel A. Tintignac ◽

Eugenia Migliavacca ◽

...

Keyword(s):

Muscle Mass ◽

Inflammatory Responses ◽

Molecular Data ◽

Model Organisms ◽

Sequencing Data ◽

Phenotypic Analysis ◽

Age Related ◽

Analogous Data ◽

Species Specific ◽

And Function

AbstractSarcopenia, the age-related loss of skeletal muscle mass and function, affects 5–13% of individuals aged over 60 years. While rodents are widely-used model organisms, which aspects of sarcopenia are recapitulated in different animal models is unknown. Here we generated a time series of phenotypic measurements and RNA sequencing data in mouse gastrocnemius muscle and analyzed them alongside analogous data from rats and humans. We found that rodents recapitulate mitochondrial changes observed in human sarcopenia, while inflammatory responses are conserved at pathway but not gene level. Perturbations in the extracellular matrix are shared by rats, while mice recapitulate changes in RNA processing and autophagy. We inferred transcription regulators of early and late transcriptome changes, which could be targeted therapeutically. Our study demonstrates that phenotypic measurements, such as muscle mass, are better indicators of muscle health than chronological age and should be considered when analyzing aging-related molecular data.

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Regulatory T Cells Protect Fine Particulate Matter-Induced Inflammatory Responses in Human Umbilical Vein Endothelial Cells

Mediators of Inflammation ◽

10.1155/2014/869148 ◽

2014 ◽

Vol 2014 ◽

pp. 1-14 ◽

Cited By ~ 6

Author(s):

Wen-cai Zhang ◽

Yan-ge Wang ◽

Zheng-feng Zhu ◽

Fang-qin Wu ◽

Yu-dong Peng ◽

...

Keyword(s):

T Cells ◽

Endothelial Cells ◽

Particulate Matter ◽

Inflammatory Cytokines ◽

Fine Particles ◽

Inflammatory Responses ◽

Umbilical Vein ◽

Fine Particulate Matter ◽

Fine Particulate ◽

Umbilical Vein Endothelial Cells

Objective. To investigate the role of CD4+CD25+T cells (Tregs) in protecting fine particulate matter (PM-) induced inflammatory responses, and its potential mechanisms.Methods. Human umbilical vein endothelial cells (HUVECs) were treated with graded concentrations (2, 5, 10, 20, and 40 µg/cm2) of suspension of fine particles for 24h. For coculture experiment, HUVECs were incubated alone, with CD4+CD25−T cells (Teff), or with Tregs in the presence of anti-CD3 monoclonal antibodies for 48 hours, and then were stimulated with or without suspension of fine particles for 24 hours. The expression of adhesion molecules and inflammatory cytokines was examined.Results. Adhesion molecules, including vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), and inflammatory cytokines, such as interleukin (IL-) 6 and IL-8, were increased in a concentration-dependent manner. Moreover, the adhesion of human acute monocytic leukemia cells (THP-1) to endothelial cells was increased and NF-κB activity was upregulated in HUVECs after treatment with fine particles. However, after Tregs treatment, fine particles-induced inflammatory responses and NF-κB activation were significantly alleviated. Transwell experiments showed that Treg-mediated suppression of HUVECs inflammatory responses impaired by fine particles required cell contact and soluble factors.Conclusions. Tregs could attenuate fine particles-induced inflammatory responses and NF-κB activation in HUVECs.

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Abstract 116: Slit-Robo Signaling Regulates Lipopolysaccharide-induced Endothelial Inflammation and Expression of Slit/Robo is Dysregulated During Endotoxemia

Circulation Research ◽

10.1161/res.113.suppl_1.a116 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Helong Zhao ◽

Appakkudal Anand ◽

Ramesh Ganju

Keyword(s):

Endothelial Cells ◽

Cell Adhesion ◽

Inflammatory Responses ◽

Umbilical Vein ◽

Model Studies ◽

Endothelial Inflammation ◽

Anti Inflammatory ◽

Whole Heart

Abstract Introduction: Lipopolysaccharide (LPS) is one of the critical factors which induce endothelial inflammation during the pathogenesis of atherosclerosis, endocarditis and sepsis shock induced heart injury. The secretory Slit2 protein and its endothelial receptors Robo1 and Robo4 have been shown to regulate mobility and permeability of endothelial cells, which could be functional in regulating LPS induced endothelial inflammation. Hypothesis: We hypothesized that in addition to regulating permeability and migration of endothelial cells, Slit2-Robo1/4 signaling might regulate other LPS-induced endothelial inflammatory responses. Methods and Results: Using Human Umbilical Vein Endothelial Cells (HUVEC) culture, we observed that Slit2 treatment suppressed LPS-induced secretion of pro-inflammatory cytokines (including GM-CSF), cell adhesion molecule upregulation and monocyte (THP-1 cell) adhesion. With siRNA knock down techniques, we further confirmed that this anti-inflammatory effect is mediated by the interaction of Slit2 with its dominant receptor in endothelial cells, Robo4, though the much lesser expressed minor receptor Robo1 is pro-inflammatory. Our signaling studies showed that downstream of Robo4, Slit2 suppressed inflammatory gene expression by inhibiting the Pyk2 - NF-kB pathway following LPS-TLR4 interaction. In addition, Slit2 can induce a positive feedback to its expression and downregulate the pro-inflammatory Robo1 receptor via mediation of miR-218. Moreover, both in in vitro studies using HUVEC and in vivo mouse model studies indicated that LPS also causes endothelial inflammation by downregulating the anti-inflammatory Slit2 and Robo4 and upregulating the pro-inflammatory Robo1 during endotoxemia, especially in mouse arterial endothelial cells and whole heart. Conclusions: Slit2-Robo1/4 signaling is important in regulation of LPS induced endothelial inflammation, and LPS in turn causes inflammation by interfering with the expression of Slit2, Robo1 and Robo4. This implies that Slit2-Robo1/4 is a key regulator of endothelial inflammation and its dysregulation during endotoxemia is a novel mechanism for LPS induced cardiovascular pathogenesis.

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Proliferation and functionality of human umbilical vein endothelial cells on angiopoietin-1 immobilized 316L stainless steel

Journal of Materials Chemistry B ◽

10.1039/c5tb01313e ◽

2015 ◽

Vol 3 (44) ◽

pp. 8717-8728 ◽

Cited By ~ 8

Author(s):

Xin Li ◽

Shuheng Yuan ◽

Si Chen ◽

Rifang Luo ◽

Kaiqin Xiong ◽

...

Keyword(s):

Stainless Steel ◽

Endothelial Cells ◽

316L Stainless Steel ◽

Umbilical Vein ◽

Human Umbilical Vein ◽

Umbilical Vein Endothelial Cells ◽

And Function ◽

Functionalized Surface ◽

Angiopoietin 1

An angiopoietin-1 functionalized surface was establishedviapolydopamine coating and regulated HUVECs survival, proliferation and function.

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Differential binding of hyaluronan on the surface of tissue-specific endothelial cell lines.

Acta Biochimica Polonica ◽

10.18388/abp.2008_3198 ◽

2008 ◽

Vol 55 (1) ◽

pp. 35-42 ◽

Cited By ~ 13

Author(s):

Karol Szczepanek ◽

Claudine Kieda ◽

Joanna Cichy

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Tumor Cells ◽

Cell Lines ◽

Tissue Specific ◽

Independent Mechanism ◽

Differential Binding ◽

And Function ◽

Pathologic Processes ◽

Tissue Origin

Tissue-specific heterogeneity of endothelial cells, both structural and functional, plays a crucial role in physiologic as well as pathologic processes, including inflammation, autoimmune diseases and tumor metastasis. This heterogeneity primarily results from the differential expression of adhesion molecules that are involved in the interactions between endothelium and circulating immune cells or disseminating tumor cells. Among these molecules present on endothelial cells is hyaluronan (HA), a glycosaminoglycan that contributes to primary (rolling) interactions through binding to its main receptor CD44 expressed on leukocytes and tumor cells. While the regulation of CD44 expression and function on either leukocytes or tumor cells has been well characterized, much less is known about the ability of endothelial cells to express HA on their surface. Therefore, in these studies we analyzed HA levels on tissue-specific endothelium. We used endothelial cell lines of different origin, including lung, skin, gut and lymph nodes that had been established previously as model lines to study interactions between the endothelium and leukocytes/tumor cells. Our results indicate that HA is accumulated on the surface of all endothelial cells examined. Moreover, retention of endogenous HA differs between the lines and may depend on their tissue origin. Analysis of binding of exogenous HA reveals the presence of specific HA binding sites on all endothelial cell lines tested. However, the retention of endogenous HA and the binding of exogenous HA is mediated through a CD44-independent mechanism.

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Abstract P069: Microvesicles From Enos-suppressed Endothelial Cells Induce Endothelial Cell Dysfunction

Hypertension ◽

10.1161/hyp.76.suppl_1.p069 ◽

2020 ◽

Vol 76 (Suppl_1) ◽

Author(s):

Vinicius P Garcia ◽

Jamie G Hijmans ◽

Kelly A Stockelman ◽

Madden Brewster ◽

Hannah Fandl ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Umbilical Vein ◽

No Production ◽

Cell Dysfunction ◽

Enos Activity ◽

Umbilical Vein Endothelial Cells ◽

And Function ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

Introduction: Endothelial nitric oxide synthase (eNOS) activity is critical to vascular health. Impaired eNOS activity and diminished NO production are common characteristics of a proatherogenic, dysfunctional endothelial phenotype that is associated with cardiovascular risk factors and disease. Extracellular microvesicles, particularly endothelial cell derived microvesicles (EMVs) represent novel mechanistic mediators of endothelial dysfunction and vascular disease. It is unknown whether eNOS suppression affects EMV number and function. We tested the following hypotheses: 1) eNOS blockade increases EMV release; and 2) EMVs derived from eNOS-suppressed cells adversely affect endothelial cell inflammation, apoptosis and NO production. Methods: Human umbilical vein endothelial cells (HUVECs) were treated with the eNOS inhibitor, L-N G -Nitroarginine methyl ester (L-NAME; 300mM) for 24 h. EMVs (CD144 + ) released into the supernatant from cells treated with L-NAME or vehicle were isolated and quantified by flow cytometry. Fresh HUVECs were then treated with either L-NAME-derived or control EMVs for 24 h. To evaluate the role of endocytosis on the endothelial effects of EMVs, HUVECs were pre-incubated (12 h) with EIPA, filipin and chlorpromazine for 2 h, and all experiments repeated. Results: EMV release was markedly higher (~100%; P<0.05) in cells treated with L-NAME compared with control (81±6 vs. 40±7 EMV/μL). L-NAME-generated EMVs induced significantly higher release of IL-6 (38.4±5.1 vs. 21.0±1.9 pg/mL) and IL-8 (38.9±3.5 vs. 27.2±3.1 pg/mL) as well as greater active NF-κB p65 (Ser-536) (9.7±0.7 vs. 6.1±0.6 AU) expression than control EMVs. The expression of activated-caspase-3 was significantly higher in the cells treated with L-NAME (9.5±1.1 vs. 6.4±0.4 AU). Total eNOS (97.1±8.2 vs. 157.5±15.6 AU), activated eNOS (4.9±1.2 vs. 9.1±1.3 AU) and NO production (5.0±0.8 vs. 7.0±0.6 μmol/L) were significantly lower in endothelial cells treated with EMVs from eNOS suppressed cells. Endocytosis blockers mitigated the deleterious endothelial effects of EMVs. Conclusion: eNOS-suppression increases EMV release. Moreover, EMVs from eNOS-suppressed cells increase endothelial cell inflammation and apoptosis and decrease NO production.

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Adhesion of flowing monocytes to hypoxia-reoxygenation-exposed endothelial cells: role of Rac1, ROS, and VCAM-1

AJP Cell Physiology ◽

10.1152/ajpcell.00301.2001 ◽

2002 ◽

Vol 283 (1) ◽

pp. C93-C102 ◽

Cited By ~ 30

Author(s):

C. K. Domingos Ng ◽

Shailesh S. Deshpande ◽

Kaikobad Irani ◽

B. Rita Alevriadou

Keyword(s):

Endothelial Cells ◽

Molecular Mechanisms ◽

Tissue Injury ◽

Umbilical Vein ◽

Ischemia Reperfusion ◽

Small Gtpase ◽

Dominant Negative ◽

Pyrrolidine Dithiocarbamate ◽

U937 Cells ◽

Hypoxia Reoxygenation

Production of reactive oxygen species (ROS) by ischemic tissue after ischemia-reperfusion (I/RP) is an important factor that contributes to tissue injury. The small GTPase Rac1 mediates the oxidative burst, and ROS act on signaling pathways involved in expression of inflammatory genes. Because there is evidence implicating monocytes in the pathogenesis of I/RP injury, our objective was to determine the molecular mechanisms that regulate adhesive interactions between monocytes and hypoxia-reoxygenation (H/RO)-exposed cultured endothelial cells (ECs). When U937 cells were perfused over human umbilical vein ECs at 1 dyn/cm2, H (1 h at 1% O2)/RO (13 h) significantly increased the fluxes of rolling and stably adherent U937 cells. Either EC treatment with the antioxidant pyrrolidine dithiocarbamate (PDTC) or infection with AdRac1N17, which results in expression of the dominant-negative form of Rac1, abolished H/RO-induced ROS production, attenuated rolling, and abolished stable adhesion of U937 cells to H/RO-exposed ECs. Infection with AdRac1N17 also abolished H/RO-induced upregulation of vascular cell adhesion molecule (VCAM)-1. In turn, blocking VCAM-1 abolished U937 cell stable adhesion and slightly increased rolling. We concluded that the Rac1-dependent ROS partially regulate rolling and exclusively regulate stable adhesion of monocytic cells to ECs after H/RO and that stable adhesion, but not rolling, is mediated by ROS-induced expression of VCAM-1.

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