scholarly journals Shiga Toxin 2 Reduces Complement Inhibitor CD59 Expression on Human Renal Tubular Epithelial and Glomerular Endothelial Cells

2013 ◽  
Vol 81 (8) ◽  
pp. 2678-2685 ◽  
Author(s):  
Silvia Ehrlenbach ◽  
Alejandra Rosales ◽  
Wilfried Posch ◽  
Doris Wilflingseder ◽  
Martin Hermann ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Shiga Toxin ◽  
Human Kidney ◽  
Enzyme Linked Immunosorbent Assay ◽  
Content Type ◽  
Protein Levels ◽  
Glomerular Endothelial Cells ◽  
Shiga Toxin 2 ◽  
Renal Tubular

ABSTRACTInfections with enterohemorrhagicEscherichia coli(EHEC) are a primary cause of hemolytic-uremic syndrome (HUS). Recently, Shiga toxin 2 (Stx2), the major virulence factor of EHEC, was reported to interact with complement, implying that the latter is involved in the pathogenesis of EHEC-induced HUS. The aim of the present study was to investigate the effect of Stx2 on the expression of membrane-bound complement regulators CD46, CD55, and CD59 on proximal tubular epithelial (HK-2) and glomerular endothelial (GEnC) cells derived from human kidney cells that are involved in HUS. Incubation with Stx2 did not influence the amount of CD46 or CD55 on the surface of HK-2 and GEnC cells, as determined by fluorescence-activated cell sorter analysis. In contrast, CD59 was significantly reduced by half on GEnC cells, but the reduction on HK-2 cells was less pronounced. With increasing amounts of Stx2, reduction of CD59 also reached significance in HK-2 cells. Enzyme-linked immunosorbent assay analyses showed that CD59 was not present in the supernatant of Stx2-treated cells, implying that CD59 reduction was not caused by cleavage from the cell surface. In fact, reverse transcription-quantitative PCR analyses showed downregulation of CD59 mRNA as the likely reason for CD59 cell surface reduction. In addition, a significant increase in terminal complement complex deposition on HK-2 cells was observed after treatment with Stx2, as a possible consequence of CD59 downregulation. In summary, Stx2 downregulates CD59 mRNA and protein levels on tubular epithelial and glomerular endothelial cells, and this downregulation likely contributes to complement activation and kidney destruction in EHEC-associated HUS.

scholarly journals The Shiga Toxin Receptor Globotriaosylceramide as Therapeutic Target in Shiga Toxin E. coli Mediated HUS

2021 ◽  
Vol 9 (10) ◽  
pp. 2157
Author(s):  
Wouter J. C. Feitz ◽  
Romy Bouwmeester ◽  
Thea J. A. M. van der Velden ◽  
Susan Goorden ◽  
Christoph Licht ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Shiga Toxin ◽  
Therapeutic Target ◽  
Human Kidney ◽  
Endothelial Damage ◽  
E Coli ◽  
Activated State ◽  
Alpha Galactosidase ◽  
Synthase Inhibitor

In 90% of the cases, childhood hemolytic uremic syndrome (HUS) is caused by an infection with the Shiga toxin (Stx) producing E. coli bacteria (STEC-HUS). Stx preferentially binds to its receptor, the glycosphingolipid, globotriaosylceramide (Gb3), present on the surface of human kidney cells and various organs. In this study, the glycosphingolipid pathway in endothelial cells was explored as therapeutic target for STEC-HUS. Primary human glomerular microvascular endothelial cells (HGMVECs) and human blood outgrowth endothelial cells (BOECs) in quiescent and activated state were pre-incubated with Eliglustat (Cerdelga®; glucosylceramide synthase inhibitor) or Agalsidase alpha (Replagal®; human cell derived alpha-galactosidase) in combination with various concentrations of Stx2a. Preincubation of endothelial cells with Agalsidase resulted in an increase of α-galactosidase activity in the cell, but had no effect on the binding of Stx to the cell surface when compared to control cells. However, the incubation of both types of endothelial cells incubated with or without the pro-inflammatory cytokine TNFα in combination with Eliglustat resulted in significant decrease of Stx binding to the cell surface, a decrease in protein synthesis by Stx2a, and diminished cellular Gb3 levels as compared to control cells. In conclusion, inhibition of the synthesis of Gb3 may be a potential future therapeutic target to protect against (further) endothelial damage caused by Stx.

scholarly journals Autoantibodies against the fibrinolytic receptor, annexin 2, in antiphospholipid syndrome

Blood ◽  
2006 ◽  
Vol 107 (11) ◽  
pp. 4375-4382 ◽  
Author(s):  
Gabriela Cesarman-Maus ◽  
Nina P. Ríos-Luna ◽  
Arunkumar B. Deora ◽  
Bihui Huang ◽  
Rosario Villa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Antiphospholipid Syndrome ◽  
Lupus Erythematosus ◽  
Antiphospholipid Antibodies ◽  
Cell Surface Receptor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Target Antigen ◽  
Healthy Individuals ◽  
Annexin 2

AbstractThe association of thrombosis and gestational morbidity with antiphospholipid antibodies is termed antiphospholipid syndrome (APS). Annexin 2 (A2) is a profibrinolytic endothelial cell surface receptor that binds plasminogen, its tissue activator (tPA), and β2-glycoprotein I (β2GPI), the main antigen for antiphospholipid antibodies. Here, we evaluate A2 as a target antigen in APS. Serum samples from 434 individuals (206 patients with systemic lupus erythematosus without thrombosis, 62 with APS, 21 with nonautoimmune thrombosis, and 145 healthy individuals) were analyzed by enzyme-linked immunosorbent assay (ELISA) and immunoblot for antiphospholipid and A2 antibodies. Anti-A2 antibodies (titer > 3 SDs) were significantly more prevalent in patients with APS (22.6%; venous, 17.5%; arterial, 34.3%; and mixed thrombosis, 40.4%) than in healthy individuals (2.1%, P < .001), patients with nonautoimmune thrombosis (0%, P = .017), or patients with lupus without thrombosis (6.3%, P < .001). Anti–A2 IgG enhanced the expression of tissue factor on endothelial cells (6.4-fold ± 0.13-fold SE), blocked A2-supported plasmin generation in a tPAdependent generation assay (19%-71%) independently of β2GPI, and inhibited cell surface plasmin generation on human umbilical vein endothelial cells (HUVECs) by 34% to 83%. We propose that anti-A2 antibodies contribute to the prothrombotic diathesis in antiphospholipid syndrome.

Differential cytotoxic actions of Shiga toxin 1 and Shiga toxin 2 on microvascular and macrovascular endothelial cells

2011 ◽  
Vol 105 (03) ◽  
pp. 515-528 ◽  
Author(s):  
Andreas Bauwens ◽  
Martina Bielaszewska ◽  
Björn Kemper ◽  
Patrik Langehanenberg ◽  
Gert von Bally ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shiga Toxin ◽  
Cell Injury ◽  
Vascular Endothelial Cells ◽  
Single Cells ◽  
Digital Holographic Microscopy ◽  
Vascular Endothelial ◽  
Enterohaemorrhagic Escherichia Coli ◽  
Shiga Toxin 2 ◽  
Comprehensive Comparison

SummaryShiga toxin (Stx)-mediated injury to vascular endothelial cells in the kidneys, brain and other organs underlies the pathogenesis of haemolytic uraemic syndrome (HUS) caused by enterohaemorrhagic Escherichia coli (EHEC). We present a direct and comprehensive comparison of cellular injury induced by the two major Stx types, Stx1 and Stx2, in human brain microvascular endothelial cells (HBMECs) and EA.hy 926 macro-vascular endothelial cells. Scanning electron microscopy of microcarrier-based cell cultures, digital holographic microscopy of living single cells, and quantitative apoptosis/necrosis assays demonstrate that Stx1 causes both necrosis and apoptosis, whereas Stx2 induces almost exclusively apoptosis in both cell lines. Moreover, microvascular and macrovascular endothelial cells have different susceptibilities to the toxins: EA.hy 926 cells are slightly, but significantly (~ 10 times) more susceptible to Stx1, whereas HBMECs are strikingly (≥ 1,000 times) more susceptible to Stx2. These findings have implications in the pathogenesis of HUS, and suggest the existence of yet to be delineated Stx type-specific mechanisms of endothelial cell injury beyond inhibition of protein bio-synthesis.

scholarly journals Shiga Toxin Selectively Upregulates Expression of Syndecan-4 and Adhesion Molecule ICAM-1 in Human Glomerular Microvascular Endothelium

Toxins ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 435
Author(s):  
Elena B. Volokhina ◽  
Wouter J. C. Feitz ◽  
Lonneke M. Elders ◽  
Thea J. A. M. van der Velden ◽  
Nicole C. A. J. van de Kar ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Adhesion Molecule ◽  
Shiga Toxin ◽  
Alternative Pathway ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Intercellular Adhesion Molecule ◽  
Intercellular Adhesion Molecule 1 ◽  
Microvascular Endothelium ◽  
Protein Levels

Hemolytic uremic syndrome (HUS) is a severe renal disease that is often preceded by infection with Shiga toxin (Stx)-producing Escherichia coli (STEC). The exact mechanism of Stx-mediated inflammation on human glomerular microvascular endothelial cells (HGMVECs) during HUS is still not well understood. In this study, we investigated the effect of Stx1 on the gene expression of proteins involved in leucocyte-mediated and complement-mediated inflammation. Our results showed that Stx1 enhances the mRNA and protein expression of heparan sulfate proteoglycan (HSPG) syndecan-4 in HGMVECs pre-stimulated with tumor necrosis factor α (TNFα). CD44 was upregulated on mRNA but not on protein level; no effect on the mRNA expression of other tested HSPGs glypican-1 and betaglycan was observed. Furthermore, Stx1 upregulated the mRNA, cell surface expression, and supernatant levels of the intercellular adhesion molecule-1 (ICAM-1) in HGMVECs. Interestingly, no effect on the protein levels of alternative pathway (AP) components was observed, although C3 mRNA was upregulated. All observed effects were much stronger in HGMVECs than in human umbilical endothelial cells (HUVECs), a common model cell type used in endothelial studies. Our results provide new insights into the role of Stx1 in the pathogenesis of HUS. Possibilities to target the overexpression of syndecan-4 and ICAM-1 for STEC-HUS therapy should be investigated in future studies.

scholarly journals Correction to: Matrix metalloproteinase 9 induces endothelial-mesenchymal transition via Notch activation in human kidney glomerular endothelial cells

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Ye Zhao ◽  
Xi Qiao ◽  
Lihua Wang ◽  
Tian Kui Tan ◽  
Hong Zhao ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Matrix Metalloproteinase ◽  
Human Kidney ◽  
Matrix Metalloproteinase 9 ◽  
Mesenchymal Transition ◽  
Glomerular Endothelial Cells ◽  
Metalloproteinase 9 ◽  
Endothelial Mesenchymal Transition ◽  
Notch Activation

An amendment to this paper has been published and can be accessed via the original article.

Impaired trafficking of distal renal tubular acidosis mutants of the human kidney anion exchanger kAE1

2002 ◽  
Vol 282 (5) ◽  
pp. F810-F820 ◽  
Author(s):  
Janne A. Quilty ◽  
Jing Li ◽  
Reinhart A. Reithmeier
Keyword(s):  
Cell Surface ◽  
Renal Tubular Acidosis ◽  
Anion Exchanger ◽  
Human Kidney ◽  
Distal Renal Tubular Acidosis ◽  
Dominant Negative ◽  
Cell Surface Expression ◽  
Dominant Negative Effect ◽  
Inherited Disease ◽  
Renal Tubular

Distal renal tubular acidosis (dRTA) is an inherited disease characterized by the failure of the kidneys to appropriately acidify urine and is associated with mutations in the anion exchanger (AE)1 gene. The effect of the R589H dRTA mutation on the expression of the human erythroid AE1 and the truncated kidney form (kAE1) was examined in transfected human embryonic kidney 293 cells. AE1, AE1 R589H, and kAE1 were present at the cell surface, whereas kAE1 R589H was located primarily intracellularly as shown by immunofluorescence, cell surface biotinylation, N-glycosylation, and anion transport assays. Coexpression of kAE1 R589H reduced the cell surface expression of kAE1 and AE1 by a dominant-negative effect, due to heterodimer formation. The mutant AE1 and kAE1 bound to an inhibitor affinity resin, suggesting that they were not grossly misfolded. Other mutations at R589 also prevented the formation of the cell surface form of kAE1, indicating that this conserved arginine residue is important for proper trafficking. The R589H dRTA mutation creates a severe trafficking defect in kAE1 but not in erythroid AE1.

Cytotoxic effect of Shiga toxin-2 holotoxin and its B subunit on human renal tubular epithelial cells

2006 ◽  
Vol 8 (2) ◽  
pp. 410-419 ◽  
Author(s):  
Virginia Pistone Creydt ◽  
Claudia Silberstein ◽  
Elsa Zotta ◽  
Cristina Ibarra
Keyword(s):  
Epithelial Cells ◽  
Shiga Toxin ◽  
Cytotoxic Effect ◽  
Tubular Epithelial Cells ◽  
Renal Tubular Epithelial Cells ◽  
B Subunit ◽  
Shiga Toxin 2 ◽  
Renal Tubular

scholarly journals Induction by Sphingomyelinase of Shiga Toxin Receptor and Shiga Toxin 2 Sensitivity in Human Microvascular Endothelial Cells

2003 ◽  
Vol 71 (2) ◽  
pp. 845-849 ◽  
Author(s):  
T. G. Obrig ◽  
R. M. Seaner ◽  
M. Bentz ◽  
C. A. Lingwood ◽  
B. Boyd ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shiga Toxin ◽  
Microvascular Endothelial Cells ◽  
Cytotoxic Action ◽  
Ceramide Level ◽  
Shiga Toxins ◽  
Shiga Toxin 2 ◽  
Toxin Receptor ◽  
Uremic Syndrome ◽  
Microvascular Endothelial

ABSTRACT Shiga toxin-producing enterohemorrhagic Escherichia coli is the major cause of acute renal failure in young children. The interaction of Shiga toxins 1 and 2 (Stx1 and Stx2) with endothelial cells is an important step in the renal coagulation and thrombosis observed in hemolytic uremic syndrome. Previous studies have shown that bacterial lipopolysaccharide and host cytokines slowly sensitize endothelial cells to Shiga toxins. In the present study, bacterial neutral sphingomyelinase (SMase) rapidly (1 h) sensitized human dermal microvascular endothelial cells (HDMEC) to the cytotoxic action of Stx2. Exposure of endothelial cells to neutral SMase (0.067 U/ml) caused a rapid increase of intracellular ceramide that persisted for hours. Closely following the change in ceramide level was an increase in the expression of globotriaosylceramide (Gb3), the receptor for Stx2. A rapid increase was also observed in the mRNA for ceramide:glucosyltransferase (CGT), the first of three glycosyltransferase enzymes of the Gb3 biosynthetic pathway. The product of CGT (glucosylceramide) was also increased. In contrast, mRNA for the third enzyme of the pathway, Gb3 synthase, was constitutively produced and was not influenced by SMase treatment of HDMEC. These results describe a rapid response mechanism by which extracellular neutral SMase derived from either bacteria or eukaryotic cells may signal endothelial cells to become sensitive to Shiga toxins.

scholarly journals MicroRNAs Regulate Cytokine Responses in Gingival Epithelial Cells

2016 ◽  
Vol 84 (12) ◽  
pp. 3282-3289 ◽  
Author(s):  
Steven C. Y. Chen ◽  
Christos Constantinides ◽  
Moritz Kebschull ◽  
Panos N. Papapanou
Keyword(s):  
Epithelial Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Periodontal Pathogens ◽  
Loss Of Function ◽  
Microbial Infection ◽  
Western Blot Assay ◽  
Content Type ◽  
Protein Levels ◽  
Gingival Epithelial Cells ◽  
Protease Deficient

MicroRNAs (miRNAs) have been established as key regulators of various biological processes with possible involvement in the pathobiology of periodontal disease. Expanding our earlier observations of substantial differential expression of specific miRNAs between clinically healthy and periodontitis-affected gingival tissues, we used miRNA inhibitors (sponges) in loss-of-function experiments to investigate the involvement of specific miRNAs in the response of pocket epithelium-derived, telomerase-immortalized human gingival keratinocytes (TIGKs) to microbial infection. We constructed stable knockdown (KD) cell lines for five epithelium-expressed miRNAs (miR-126, miR-141, miR-155, miR-210, and miR-1246) and assessed their response to infection with periodontal pathogens using microarray analysis, quantitative PCR (qPCR), enzyme-linked immunosorbent assay (ELISA), and Western blot assay. miR-126 KD cells showed lower expression of interleukin 8 (IL-8) and CXCL1, both on the mRNA and protein levels, than did controls upon stimulation by heat-killed wild-typePorphyromonas gingivalis, liveP. gingivalisprotease-deficient mutant KDP128, and liveAggregatibacter actinomycetemcomitans. In contrast, infection of miR-155 KD and miR-210 KD cells with the same organisms resulted in higher IL-8 and CXCL1 mRNA and protein expression. These effects appeared to be regulated by NF-κB, as suggested by altered transcription and/or phosphorylation status of components of the NF-κB system. Reduced neutrophil-like HL-60 cell chemotactic activity was observed in response to infection of miR-126 KD cells, indicating that miR-126 plays an important role in immune responses. Our findings indicate that specific miRNAs regulate the expression of inflammatory cytokines in human gingival epithelial cells in response to microbial infection.

scholarly journals Attenuation of Glomerular Endothelial Cells from High Glucose-Induced Injury by Blockade of MAD2B

2015 ◽  
Vol 35 (1) ◽  
pp. 61-70 ◽  
Author(s):  
Yu-Mei Wang ◽  
Yu Hao ◽  
Xian-Fang Meng ◽  
Fang-Fang He ◽  
Shan Chen ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
High Glucose ◽  
Cell Monolayer ◽  
No Production ◽  
Protein Levels ◽  
Glomerular Endothelial Cells ◽  
Monolayer Permeability ◽  
Glucose Treatment ◽  
In Cells

Background/Aims: To assess the role of mitotic arrest-deficient 2-like protein 2 (MAD2B) in high glucose-induced injury in mouse glomerular endothelial cells (GEnCs). Methods: GEnCs were cultured in vitro, and MAD2B protein levels were measured by Western blot in cells stimulated with high glucose (30 mM) for various periods of time. MAD2B and scrambled shRNA were introduced into GEnCs by liposomal transfection. Cell proliferation, apoptosis, nitric oxide (NO) production, and monolayer permeability were then measured in cells grown in the following conditions: control, high glucose treatment, MAD2B shRNA transfection with high glucose treatment, and scrambled shRNA transfection with high glucose treatment. Results: High glucose increased the protein levels of MAD2B in GEnCs. Compared with control cells, apoptosis was increased by high glucose treatment, which was attenuated by transfection with MAD2B shRNA transfection. Cells treated with high glucose produced less NO than control cells, whereas MAD2B shRNA transfection increased NO production. Cell monolayer permeability was enhanced in high glucose treated cells, but MAD2B shRNA transfection reduced permeability. Conclusion: High glucose levels induced the expression of MAD2B in GEnCs, whereas suppressing its expression reduced high glucose-induced endothelial cell apoptosis and high permeability, and promoted cell proliferation and NO production.

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