Primary Human Derived Blood Outgrowth Endothelial Cells: An Appropriate In Vitro Model to Study Shiga Toxin Mediated Damage of Endothelial Cells

Hemolytic uremic syndrome (HUS) is a rare disease primarily characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. Endothelial damage is the hallmark of the pathogenesis of HUS with an infection with the Shiga toxin (Stx) producing Escherichia coli (STEC-HUS) as the main underlying cause in childhood. In this study, blood outgrowth endothelial cells (BOECs) were isolated from healthy donors serving as controls and patients recovered from STEC-HUS. We hypothesized that Stx is more cytotoxic for STEC-HUS BOECs compared to healthy donor control BOECs explained via a higher amount of Stx bound to the cell surface. Binding of Shiga toxin-2a (Stx2a) was investigated and the effect on cytotoxicity, protein synthesis, wound healing, and cell proliferation was studied in static conditions. Results show that BOECs are highly susceptible for Stx2a. Stx2a is able to bind to the cell surface of BOECs with cytotoxicity in a dose-dependent manner as a result. Pre-treatment with tumor necrosis factor alpha (TNF-α) results in enhanced Stx binding with 20–30% increased lactate dehydrogenase (LDH) release. Endothelial wound healing is delayed in a Stx2a-rich environment; however, this is not caused by an effect on the proliferation rate of BOECs. No significant differences were found between control BOECs and BOECs from recovered STEC-HUS patients in terms of Stx2a binding and inhibition of protein synthesis.

Download Full-text

Cell Biological Responses after Shiga Toxin-1 Exposure to Primary Human Glomerular Microvascular Endothelial Cells from Pediatric and Adult Origin

International Journal of Molecular Sciences ◽

10.3390/ijms22115615 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5615

Author(s):

Wouter J. C. Feitz ◽

Petra A. van Setten ◽

Thea J. A. M. van der Velden ◽

Christoph Licht ◽

Lambert P. J. W. van den Heuvel ◽

...

Keyword(s):

Endothelial Cells ◽

Protein Synthesis ◽

Shiga Toxin ◽

Age Groups ◽

Kidney Tissue ◽

Microvascular Endothelial Cells ◽

Dependent Manner ◽

Biological Responses ◽

Microvascular Endothelial

Hemolytic uremic syndrome (HUS) is characterized by a triad of symptoms consisting of hemolytic anemia, thrombocytopenia and acute renal failure. The most common form of HUS is caused by an infection with Shiga toxin (Stx) producing Escherichia coli bacteria (STEC-HUS), and the kidneys are the major organs affected. The development of HUS after an infection with Stx occurs most frequently in children under the age of 5 years. However, the cause for the higher incidence of STEC-HUS in children compared to adults is still not well understood. Human glomerular microvascular endothelial cells (HGMVECs) isolated and cultured from pediatric and adult kidney tissue were investigated with respect to Stx binding and different cellular responses. Shiga toxin-1 (Stx-1) inhibited protein synthesis in both pediatric and adult HGMVECs in a dose-dependent manner at basal conditions. The preincubation of pediatric and adult HGMVECs for 24 hrs with TNFα resulted in increased Stx binding to the cell surface and a 20–40% increase in protein synthesis inhibition in both age groups. A decreased proliferation of cells was found when a bromodeoxyuridine (BrdU) assay was performed. A trend towards a delay in endothelial wound closure was visible when pediatric and adult HGMVECs were incubated with Stx-1. Although minor differences between pediatric HGMVECs and adult HGMVECs were found in the assays applied in this study, no significant differences were observed. In conclusion, we have demonstrated that in vitro primary HGMVECs isolated from pediatric and adult kidneys do not significantly differ in their cell biological responses to Stx-1.

Download Full-text

TNFɑ-induced LDL cholesterol accumulation involve elevated LDLR cell surface levels and SR-B1 downregulation in human arterial endothelial cells

10.21203/rs.3.rs-267515/v1 ◽

2021 ◽

Author(s):

Emmanuel Ugochukwu Okoro

Keyword(s):

Endothelial Cells ◽

Cell Surface ◽

Ldl Cholesterol ◽

Oxidized Ldl ◽

Scavenger Receptor ◽

Density Lipoprotein ◽

Surface Proteins ◽

Surface Binding ◽

Necrosis Factor Alpha ◽

Arterial Endothelial Cells

Abstract Background Excess lipid droplets are frequently observed in arterial endothelial cells at sites of advanced atherosclerotic plaques. The mechanism or implication of this observation is unknown. Methods The role of tumor necrosis factor alpha (TNFɑ) in modulating low density lipoprotein (LDL) content in confluent primary human aortic endothelial cells (pHAECs) was investigated. Results TNFɑ promoted up to 2 folds increase in cellular cholesterol, which was resistant to ACAT inhibition. The cholesterol increase was associated with increased 125I-LDL surface binding. Using the non-hydrolysable label, Dil, TNFɑ could induce a massive increase in Dil-LDL by over 200 folds. The elevated intracellular Dil-LDL was blocked with excess unlabeled LDL and PCSK9, but not oxidized LDL (oxLDL), RAP, or apoE depletion. Moreover, TNFɑ-induced increase of LDL-derived lipids was elevated through lysosome inhibition. Using specific LDLR antibody, the Dil-LDL accumulation was reduced by over 99%. Effects of TNFɑ included LDLR cell surface increase by 138%, and very large increases in ICAM-1 total and surface proteins, respectively. In contrast, that of scavenger receptor B1 (SR-B1) was reduced. Additionally, LDLR antibody bound rapidly in TNFɑ-treated cells by about 30 folds, inducing a migrating shift in the LDLR protein. The effect of TNFɑ on Dil-LDL accumulation was inhibited by the antioxidant tetramethythiourea (TMTU) dose-dependently, but not by downstream cascade inhibitors against NF-κB, stress kinases, ASK1, JNK, p38, or apoptosis caspases. Grown on transwell inserts, TNFɑ did not enhance apical (AP) to basolateral (BL) LDL cholesterol or Dil release. Conclusions It is concluded that TNFɑ promotes LDLR functions through combined increase at the cell surface and SR-B1 downregulation. This may have important implications on progression of atherosclerosis.

Download Full-text

Angiotensin II increases the release of endothelin-I from human cultured endothelial cells but does not regulate its circulating levels

Clinical Science ◽

10.1042/cs0960261 ◽

1999 ◽

Vol 96 (3) ◽

pp. 261-270 ◽

Cited By ~ 1

Author(s):

Claudio FERRI ◽

Giovambattista DESIDERI ◽

Roberta BALDONCINI ◽

Cesare BELLINI ◽

Marco VALENTI ◽

...

Keyword(s):

Endothelial Cells ◽

Protein Synthesis ◽

Angiotensin Ii ◽

At1 Receptor ◽

Dependent Manner ◽

Synthesis Inhibitor ◽

Endothelin 1 ◽

Cultured Endothelial Cells

We investigated the effect of angiotensin II on endothelin-1 secretion in vitro and in vivo. In vivo, angiotensin II was given intravenously to 23 essential hypertensive and 8 control subjects according to different protocols: Study A, 1.0 ngċmin-1ċkg-1 and 3.0 ngċmin-1ċkg-1 angiotensin II for 30 min each; Study B, 1.0 ngċmin-1ċkg-1 and 3.0 ngċmin-1ċkg-1 angiotensin II for 120 min each; Study C, 3.0 ngċmin-1ċkg-1 angiotensin II for 30 min followed by a dose increment of 3.0 ngċmin-1ċkg-1 every 30 min until mean blood pressure levels increased by 25 mmHg; Study D, 1.0 ngċmin-1ċkg-1 followed by 3.0 ngċmin-1ċkg-1 angiotensin II for 60 min each on two different NaCl diets (either 20 mmol NaCl/day or 220 mmol NaCl/day, both for 1 week). In all in vivo studies neither plasma nor urine endothelin-1 levels changed with angiotensin II infusion. In contrast, angiotensin II (10-9, 10-8, 10-7 mol/l) stimulated endothelin-1 secretion from cultured human vascular endothelial cells derived from umbilical cord veins in a time- and dose-dependent manner. The in vitro angiotensin II effects were abolished by candesartan cilexetil, an inhibitor of the membrane-bound AT1 receptor, and also by actinomycin D, an RNA synthesis inhibitor, and cycloheximide, a protein synthesis inhibitor, indicating that endothelin-1 release depended on AT1 receptor subtype and de novo protein synthesis. Our findings indicate that angiotensin II regulates endothelin-1 release by cultured endothelial cells through an AT1 receptor-dependent pathway, but does not influence circulating endothelin-1 levels in vivo.

Download Full-text

Bemiparin and Fluid Flow Modulate the Expression, Activity and Release of Tissue Factor Pathway Inhibitor in Human Endothelial Cells In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0037-1616761 ◽

2001 ◽

Vol 86 (12) ◽

pp. 1547-1554 ◽

Cited By ~ 13

Author(s):

Andrew Westmuckett ◽

Vijay Kakkar ◽

Tsutomu Hamuro ◽

Florea Lupu ◽

Cristina Lupu

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Cell Surface ◽

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Arterial Flow ◽

Immunogold Electron Microscopy ◽

Tissue Factor Pathway ◽

Static Conditions

SummaryWe investigated the localisation, gene expression, and activity of tissue factor pathway inhibitor (TFPI) in endothelial cells (EC) grown in static conditions or under shear stress, in the presence of unfractionated heparin (UFH) and two low-molecular-weight heparins (LMWHs), dalteparin and bemiparin (a second generation of LMWHs). All three preparations induced increased release, cellular redistribution, and enhanced activity of TFPI on the cell surface in static EC. In EC grown under shear stress (0.27, 4.1 and 19 dyne/cm2) and incubated with each heparin for 24 h, the release of TFPI was significantly correlated with the level of flow for bemiparin and dalteparin, but not for UFH. For all three levels of flow tested, bemiparin induced the highest secretion and increase of both cellular TFPI and cell surface activity of the inhibitor. The expression of TFPI mRNA, determined by Northern blotting, was specifically modulated by heparins. All three preparations increased the expression of TFPI by 60 to 120% in EC under minimal flow, but only bemiparin enhanced TFPI mRNA in EC under the arterial flow. Immunogold electron microscopy revealed that EC exhibited strong cellular labelling for TFPI when grown under arterial flow in the presence of bemiparin. We conclude that in EC subjected to shear stress in vitro bemiparin is more efficient than UFH or dalteparin in modulating the expression, release and activity of TFPI. We therefore suggest that bemiparin may be superior over the conventional heparins in maintaining the anticoagulant properties of the endothelium.

Download Full-text

TNFɑ-induced LDL cholesterol accumulation involve elevated LDLR cell surface levels and SR-B1 downregulation in human arterial endothelial cells

10.21203/rs.3.rs-267515/v2 ◽

2021 ◽

Author(s):

Emmanuel Ugochukwu Okoro

Keyword(s):

Endothelial Cells ◽

Cell Surface ◽

Ldl Cholesterol ◽

Oxidized Ldl ◽

Scavenger Receptor ◽

Density Lipoprotein ◽

Surface Proteins ◽

Surface Binding ◽

Necrosis Factor Alpha ◽

Arterial Endothelial Cells

Abstract Excess lipid droplets are frequently observed in arterial endothelial cells at sites of advanced atherosclerotic plaques. Here, the role of tumor necrosis factor alpha (TNFɑ) in modulating low density lipoprotein (LDL) content in confluent primary human aortic endothelial cells (pHAECs) was investigated. TNFɑ promoted up to 2 folds increase in cellular cholesterol, which was resistant to ACAT inhibition. The cholesterol increase was associated with increased 125I-LDL surface binding. Using the non-hydrolysable label, Dil, TNFɑ could induce a massive increase in Dil-LDL by over 200 folds. The elevated intracellular Dil-LDL was blocked with excess unlabeled LDL and PCSK9, but not oxidized LDL (oxLDL), receptor associated protein (RAP), or apolipoprotein (apoE) depletion. Moreover, TNFɑ-induced increase of LDL-derived lipids was elevated through lysosome inhibition. Using specific LDLR antibody, the Dil-LDL accumulation was reduced by over 99%. Effects of TNFɑ included LDLR cell surface increase by 138%, and very large increases in ICAM-1 total and surface proteins, respectively. In contrast, that of scavenger receptor B1 (SR-B1) was reduced. Additionally, LDLR antibody bound rapidly in TNFɑ-treated cells by about 30 folds, inducing a migrating shift in the LDLR protein. The effect of TNFɑ on Dil-LDL accumulation was inhibited by the antioxidant tetramethythiourea (TMTU) dose-dependently, but not by inhibitors against NF-κB, stress kinases, ASK1, JNK, p38, or apoptosis caspases. Grown on transwell inserts, TNFɑ did not enhance apical to basolateral LDL cholesterol or Dil release. It is concluded that TNFɑ promotes LDLR functions through combined increase at the cell surface and SR-B1 downregulation.

Download Full-text

Chronic expression of P-selectin on endothelial cells stimulated by the T-cell cytokine, interleukin-3

Blood ◽

10.1182/blood.v87.4.1432.bloodjournal8741432 ◽

1996 ◽

Vol 87 (4) ◽

pp. 1432-1438 ◽

Cited By ~ 59

Author(s):

Y Khew-Goodall ◽

CM Butcher ◽

MS Litwin ◽

S Newlands ◽

EI Korpelainen ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Surface ◽

Chronic Inflammation ◽

T Lymphocyte ◽

Mrna Level ◽

Cell Surface Expression ◽

Mutant Form ◽

Necrosis Factor Alpha ◽

Interleukin 3

P-selectin expressed on the surface of endothelium mediates leukocyte adhesion in vitro and rolling in vivo. Several inducers of cell-surface P-selectin expression on endothelial cells (EC) have previously been identified, all of which yield transient cell-surface expression of P- selectin lasting minutes to a few hours. We now show that a T- lymphocyte product, interleukin-3 (IL-3), stimulates the long-term endothelial cells (HUVEC). IL-3 induced cell-surface P-selectin expression in two phases. An initial peak at 10 minutes was followed by a prolonged upregulation beginning 16 hours after IL-3 addition and lasting at least 4 days. The level of P-selectin expression induced by IL-3 added for 48 hours was similar to that induced by treatment of HUVEC for 10 minutes with thrombin, and the effect of adding IL-3 for 48 hours followed by thrombin for 10 minutes was additive. Induction of cell-surface P-selectin expression by IL-3 was blocked by pretreatment of EC with a blocking monoclonal antibody against the IL-3 receptor alpha-chain. Lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF alpha) and a mutant form of IL-3 with decreased potency did not induce cell-surface P-selectin expression after 48 hours' incubation with HUVEC, suggesting that the effect was specific to IL-3. The increase in cell-surface P-selectin expression occurring after 16 hours of incubation with IL-3 was accompanied by a similar prolonged increase in the steady-state mRNA level that was not observed at 10 minutes after IL-3 addition. As T-lymphocyte infiltration is a hallmark of chronic inflammation, our observations suggest that the secretion of IL-3 by T lymphocytes may serve to maintain the inflammatory state during chronic inflammation.

Download Full-text

TNFɑ-Induced LDL Cholesterol Accumulation Involve Elevated LDLR Cell Surface Levels and SR-B1 Downregulation in Human Arterial Endothelial Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22126236 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6236

Author(s):

Emmanuel Ugochukwu Okoro

Keyword(s):

Endothelial Cells ◽

Cell Surface ◽

Ldl Cholesterol ◽

Oxidized Ldl ◽

Scavenger Receptor ◽

Density Lipoprotein ◽

Surface Proteins ◽

Surface Binding ◽

Necrosis Factor Alpha ◽

Arterial Endothelial Cells

Excess lipid droplets are frequently observed in arterial endothelial cells at sites of advanced atherosclerotic plaques. Here, the role of tumor necrosis factor alpha (TNFɑ) in modulating the low-density lipoprotein (LDL) content in confluent primary human aortic endothelial cells (pHAECs) was investigated. TNFɑ promoted an up to 2 folds increase in cellular cholesterol, which was resistant to ACAT inhibition. The cholesterol increase was associated with increased 125I-LDL surface binding. Using the non-hydrolysable label, Dil, TNFɑ could induce a massive increase in Dil-LDL by over 200 folds. The elevated intracellular Dil-LDL was blocked with excess unlabeled LDL and PCSK9, but not oxidized LDL (oxLDL), or apolipoprotein (apoE) depletion. Moreover, the TNFɑ-induced increase of LDL-derived lipids was elevated through lysosome inhibition. Using specific LDLR antibody, the Dil-LDL accumulation was reduced by over 99%. The effects of TNFɑ included an LDLR cell surface increase of 138%, and very large increases in ICAM-1 total and surface proteins, respectively. In contrast, that of scavenger receptor B1 (SR-B1) was reduced. Additionally, LDLR antibody bound rapidly in TNFɑ-treated cells by about 30 folds, inducing a migrating shift in the LDLR protein. The effect of TNFɑ on Dil-LDL accumulation was inhibited by the antioxidant tetramethythiourea (TMTU) dose-dependently, but not by inhibitors against NF-κB, stress kinases, ASK1, JNK, p38, or apoptosis caspases. Grown on Transwell inserts, TNFɑ did not enhance apical to basolateral LDL cholesterol or Dil release. It is concluded that TNFɑ promotes LDLR functions through combined increase at the cell surface and SR-B1 downregulation.

Download Full-text

TNFɑ-induced LDL cholesterol accumulation involve elevated LDLR cell surface levels and SR-B1 downregulation in human arterial endothelial cells

10.21203/rs.3.rs-267515/v3 ◽

2021 ◽

Author(s):

Emmanuel Ugochukwu Okoro

Keyword(s):

Endothelial Cells ◽

Cell Surface ◽

Ldl Cholesterol ◽

Oxidized Ldl ◽

Scavenger Receptor ◽

Density Lipoprotein ◽

Surface Proteins ◽

Surface Binding ◽

Necrosis Factor Alpha ◽

Arterial Endothelial Cells

Download Full-text

The pleiotropic molecule NGF regulates the in vitro properties of fibroblasts, keratinocytes, and endothelial cells: implications for wound healing

AJP Cell Physiology ◽

10.1152/ajpcell.00180.2019 ◽

2020 ◽

Vol 318 (2) ◽

pp. C360-C371 ◽

Cited By ~ 3

Author(s):

N. Gostynska ◽

M. Pannella ◽

M. L. Rocco ◽

L. Giardino ◽

L. Aloe ◽

...

Keyword(s):

Wound Healing ◽

Endothelial Cells ◽

Cell Line ◽

Biological Effects ◽

Cell Types ◽

Dependent Manner ◽

Skin Wound Healing ◽

Keratinocyte Proliferation ◽

Fibroblast Migration

Nerve growth factor (NGF) is recognized as a pleiotropic molecule, exerting a variety of biological effects on different cell types and pathophysiological conditions, and its role in tissue wound healing has been recently highlighted. However, the preferential cellular target of NGF is still elusive in the complex cellular and molecular cross talk that accompanies wound healing. Thus, to explore possible NGF cellular targets in skin wound healing, we investigated the in vitro NGF responsiveness of keratinocytes (cell line HEKa), fibroblasts (cell line BJ), and endothelial cells (cell line HUVEC), also in the presence of adverse microenvironmental conditions, e.g., hyperglycemia. The main results are summarized as follows: 1) NGF stimulates keratinocyte proliferation and HUVEC proliferation and angiogenesis in a dose-dependent manner although it has no effect on fibroblast proliferation; 2) NGF stimulates keratinocyte but not fibroblast migration in the wound healing assay; and 3) NGF completely reverts the proliferation impairment of keratinocytes and the angiogenesis impairment of HUVECs induced by high d-glucose concentration in the culture medium. These results contribute to better understanding possible targets for the therapeutic use of NGF in skin repair.

Download Full-text

Morphilogy and Ultrastructure of in Vitro Cultures of Aortic Endothelial Cells Interacting with Antibodies

10.1055/s-0039-1684767 ◽

1979 ◽

Author(s):

S. Korach ◽

D. Ngo

Keyword(s):

Endothelial Cells ◽

Cell Surface ◽

Vascular Endothelial Cells ◽

Intercellular Junctions ◽

Cell Types ◽

Endothelial Damage ◽

Antibody Concentration ◽

High Yields ◽

Scanning Em

Adult pig aortas, sectioned longitudinally, were incubated in 0.1% collagenase-PBS (15 mn, 37°C). Gentle scraping of the lumenal surface resulted in high yields (3-4 x 106 cell/aorta) of viable endothelial cells, essentially devoid of other cell types by morphological and immunochemical (F VIII-antigen) criteria. Confluent monolayers were incubated for various times (5 mn to 1 wk) with decomplemented rabbit antisera raised against pig endothelial cells. Changes in cell morphology appeared to depend on antibody concentration rather than on duration of contact with antiserum. High concentrations of antiserum (5 to 20%) led to cytoplasmic shredding, bulging of cells and extensive vacuolization, whereas at lower concentrations, cells appeared almost normal. Transmission EM studies by the indirect immunoperoxydase method showed antibodies reacting with unfixed cells to be distributed all over the upper cell surface, in the outer parts of intercellular junctions, and within numerous pinocytotic vesicles. Much weaker reactions could also be seen at the lower cell surface. When viewed under the Scanning EM, antiserum-treated endothelial cells also disclosed antibody concentration-dependent bulging and release of cells from their substrate. In vitro studies of gradual modifications of vascular endothelial cells acted upon by antibodies should provide a better understanding of the structural and biochemical processes underlying endothelial damage and detachment.

Download Full-text