scholarly journals Crosstalk between Human Microvascular Endothelial Cells and Tubular Epithelial Cells Modulates Pro-Inflammatory Responses Induced by Shiga Toxin Type 2 and Subtilase Cytotoxin

Toxins ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 648 ◽  
Author(s):  
Romina S. Álvarez ◽  
Carolina Jancic ◽  
Nicolás Garimano ◽  
Flavia Sacerdoti ◽  
Adrienne W. Paton ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Epithelial Cells ◽  
Shiga Toxin ◽  
Inflammatory Responses ◽  
Cytokine Secretion ◽  
Microvascular Endothelial Cells ◽  
Tubular Epithelial Cells ◽  
Proximal Tubular Epithelial Cells ◽  
Microvascular Endothelial ◽  
Subtilase Cytotoxin

Hemolytic uremic syndrome (HUS) is a consequence of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection and is the most frequent cause of acute renal failure (ARF) in children. Subtilase cytotoxin (SubAB) has also been associated with HUS pathogenesis. We previously reported that Stx2 and SubAB cause different effects on co-cultures of human renal microvascular endothelial cells (HGEC) and human proximal tubular epithelial cells (HK-2) relative to HGEC and HK-2 monocultures. In this work we have analyzed the secretion of pro-inflammatory cytokines by co-cultures compared to monocultures exposed or not to Stx2, SubAB, and Stx2+SubAB. Under basal conditions, IL-6, IL-8 and TNF-α secretion was different between monocultures and co-cultures. After toxin treatments, high concentrations of Stx2 and SubAB decreased cytokine secretion by HGEC monocultures, but in contrast, low toxin concentrations increased their release. Toxins did not modulate the cytokine secretion by HK-2 monocultures, but increased their release in the HK-2 co-culture compartment. In addition, HK-2 monocultures were stimulated to release IL-8 after incubation with HGEC conditioned media. Finally, Stx2 and SubAB were detected in HGEC and HK-2 cells from the co-cultures. This work describes, for the first time, the inflammatory responses induced by Stx2 and SubAB, in a crosstalk model of renal endothelial and epithelial cells.

scholarly journals Action of Shiga Toxin Type-2 and Subtilase Cytotoxin on Human Microvascular Endothelial Cells

PLoS ONE ◽  
2013 ◽  
Vol 8 (7) ◽  
pp. e70431 ◽  
Author(s):  
María M. Amaral ◽  
Flavia Sacerdoti ◽  
Carolina Jancic ◽  
Horacio A. Repetto ◽  
Adrienne W. Paton ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shiga Toxin ◽  
Microvascular Endothelial Cells ◽  
Microvascular Endothelial ◽  
Subtilase Cytotoxin

Influence of microvascular endothelial cells on transcriptional regulation of proximal tubular epithelial cells

2008 ◽  
Vol 294 (2) ◽  
pp. C543-C554 ◽  
Author(s):  
Sonia Aydin ◽  
Sara Signorelli ◽  
Thomas Lechleitner ◽  
Michael Joannidis ◽  
Clara Pleban ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Epithelial Cell ◽  
Cell Line ◽  
Cell Function ◽  
Microvascular Endothelial Cells ◽  
Tubular Epithelial Cells ◽  
Proximal Tubular Epithelial Cells ◽  
Proximal Tubular ◽  
Microvascular Endothelial

In the renal cortex the peritubular capillary network and the proximal tubular epithelium cooperate in solute and water reabsorption, secretion, and inflammation. However, the mechanisms by which these two cell types coordinate such diverse functions remain to be characterized. Here we investigated the influence of microvascular endothelial cells on proximal tubule cells, using a filter-based, noncontact, close-proximity coculture of the human microvascular endothelial cell line HMEC-1 and the human proximal tubular epithelial cell line HK-2. With the use of DNA microarrays the transcriptomes of HK-2 cells cultured in mono- and coculture were compared. HK-2 cells in coculture exhibited a differential expression of 99 genes involved in pathways such as extracellular matrix (e.g., lysyl oxidase), cell-cell communication (e.g., IL-6 and IL-1β), and transport (e.g., GLUT3 and lipocalin 2). HK-2 cells also exhibited an enhanced paracellular gating function in coculture, which was dependent on HMEC-1-derived extracellular matrix. We identified a number of HMEC-1-enriched genes that are potential regulators of epithelial cell function such as extracellular matrix proteins (e.g., collagen I, III, IV, and V, laminin-α IV) and cytokines/growth factors (e.g., hepatocyte growth factor, endothelin-1, VEGF-C). This study demonstrates a complex network of communication between microvascular endothelial cells and proximal tubular epithelial cells that ultimately affects proximal tubular cell function. This coculture model and the data described will be important in the further elucidation of microvascular endothelial and proximal tubular epithelial cross talk mechanisms.

scholarly journals Induction of the S100 Chemotactic Protein, CP-10, in Murine Microvascular Endothelial Cells by Proinflammatory Stimuli

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4812-4821 ◽  
Author(s):  
Tina Yen ◽  
Craig A. Harrison ◽  
Jannine M. Devery ◽  
Sharon Leong ◽  
Siiri E. Iismaa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Inflammatory Responses ◽  
S100 Protein ◽  
Calcium Ionophore ◽  
Delayed Type Hypersensitivity ◽  
Microvascular Endothelial Cells ◽  
Mrna Induction ◽  
Chemotactic Protein ◽  
Microvascular Endothelial

Abstract Microvascular endothelial cells (EC) have multiple functions in inflammatory responses, including the production of chemoattractants that enhance leukocyte transmigration into tissues. Chemotactic protein, 10 kD (CP-10), is an S100 protein with potent chemotactic activity for myeloid cells in vitro and in vivo and is expressed in neutrophils and lipopolysaccharide (LPS)-activated macrophages. We show here that CP-10 is induced in murine endothelioma cell lines (bEnd-3, sEnd-1, and tEnd-1) after activation with LPS and interleukin-1 (IL-1) but not tumor necrosis factor α (TNFα) or interferon γ (IFNγ). Induction was not mediated by endogenous release of IL-1 or TNFα and was not directly upregulated by phorbol myristate acetate, calcium ionophore, or vitamin D3. EC were exquisitely sensitive to IL-1 activation (3.4 U/mL) and CP-10 mRNA induction with IL-1 occurred earlier (8 hours) than with LPS (12 hours). Furthermore, some microvessels and capillaries in delayed-type hypersensitivity lesions expressed cytoplasmic CP-10. Responses to LPS and not IL-1 in vitro were regulated by the degree of cell confluence and by TNFα costimulation. The related MRP-14 mRNA had a different induction pattern. Monomeric and homodimeric CP-10 upregulated by activation was predominantly cell-associated. EC-derived CP-10 may contribute to amplification of inflammatory processes by enhancing leukocyte shape changes and transmigration in the microcirculation.

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.

Active Site-Inactivated Recombinant Factor VIIa Blocks Shiga Toxin-Induced Upregulation of Tissue Factor Activity on Human Glomerular Endothelial Cells and Human Proximal Tubular Epithelial Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 532-532
Author(s):  
Eirini Nestoridi ◽  
Rafail Kushak ◽  
Julie R. Ingelfinger ◽  
Eric F. Grabowski
Keyword(s):  
Endothelial Cells ◽  
Shiga Toxin ◽  
Active Site ◽  
Recombinant Factor Viia ◽  
Factor Viia ◽  
Tnf Alpha ◽  
Tubular Epithelial Cells ◽  
Proximal Tubular Epithelial Cells ◽  
Proximal Tubular ◽  
Human Glomerular Endothelial Cells

Abstract Post-diarrheal hemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children, is most often caused by Shiga toxin (Stx)-producing Escherichia coli infection. The most widely recognized tissue damage in HUS occurs within the kidney, most prominently with local thrombosis and platelet-fibrin accumulation, suggesting activation of the coagulation cascade. We have shown that human proximal tubular epithelial cells (HK-2 cells) exposed to Stx-1 augment their constitutive surface tissue factor (TF) activity by 3.2-fold, and that Stx-1 treatment following TNF-alpha exposure enhances TF activity by 2.7-fold on human glomerular endothelial cells (HGECs). We investigated the possibility of modulating this cellular response to Stx-1 by blocking the TF pathway with active site-inactivated recombinant factor VIIa (irFVIIa), which retains the ability to bind TF, but is enzymatically inactive, thereby inhibiting thrombin formation. Moreover, its action is limited to exposed, functional TF and does not significantly prolong the bleeding time in vivo. Monolayers of both HK-2 cells and TNF-alpha-activated HGECs were incubated at 37°C with Stx-1. Functional TF was determined chromogenically as the production rate of factor Xa. The use of 60 nM irFVIIa completely abrogated the enhanced activity of TF caused by 4 hours incubation with 10 pM Stx-1 of TNF-alpha-activated HGECs (N = 4; Figure 1). On HK-2 cells, irFVIIa (60 – 200 nM) also effectively reversed the observed TF upregulation after exposure to 1 nM Stx-1 for 22 hours as well as blocked the constitutive TF expression which is seen with this cell type (N = 3; Figure 2). The TF pathway may prove to be of major importance in the pathogenesis of HUS, and direct targeting of this pathway with irFVIIa might provide a novel therapeutic approach to this disease.

scholarly journals Anaplasma phagocytophilum-Borrelia burgdorferi Coinfection Enhances Chemokine, Cytokine, and Matrix Metalloprotease Expression by Human Brain Microvascular Endothelial Cells

2007 ◽  
Vol 14 (11) ◽  
pp. 1420-1424 ◽  
Author(s):  
Dennis J. Grab ◽  
Elvis Nyarko ◽  
Nicole C. Barat ◽  
Olga V. Nikolskaia ◽  
J. Stephen Dumler
Keyword(s):  
Endothelial Cells ◽  
Borrelia Burgdorferi ◽  
Human Brain ◽  
Anaplasma Phagocytophilum ◽  
Inflammatory Responses ◽  
Matrix Metalloproteases ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Necrosis Factor Alpha ◽  
Microvascular Endothelial

ABSTRACT Borrelia burgdorferi and Anaplasma phagocytophilum coinfect and are transmitted by Ixodes species ticks. Clinical indicators suggest that A. phagocytophilum coinfection contributes to the severity, dissemination, and, possibly, sequelae of Lyme disease. Previous in vitro studies showed that spirochete penetration through human brain microvascular endothelial cells of the blood-brain barrier is facilitated by endothelial cell-derived matrix metalloproteases (MMPs). A. phagocytophilum-infected neutrophils continuously release MMPs and other vasoactive biomediators. We examined B. burgdorferi infection of brain microvascular barriers during A. phagocytophilum coinfection and showed that coinfection enhanced reductions in transendothelial electrical resistance and enhanced or synergistically increased production of MMPs (MMP-1, -3, -7, -8, and -9), cytokines (interleukin 6 [IL-6], IL-10, and tumor necrosis factor alpha), and chemokines (IL-8 and macrophage inflammatory protein 1α) known to affect vascular permeability and inflammatory responses.

scholarly journals Insulin and IGF1 receptors in human cardiac microvascular endothelial cells: metabolic, mitogenic and anti-inflammatory effects

2012 ◽  
Vol 215 (1) ◽  
pp. 89-96 ◽  
Author(s):  
Karolina Bäck ◽  
Rakibul Islam ◽  
Git S Johansson ◽  
Simona I Chisalita ◽  
Hans J Arnqvist
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Inflammatory Responses ◽  
Receptor Protein ◽  
Microvascular Endothelial Cells ◽  
Tnf Α ◽  
Glucose Accumulation ◽  
Anti Inflammatory ◽  
Microvascular Endothelial ◽  
Cardiac Microvascular Endothelial Cells

Diabetes is associated with microcirculatory dysfunction and heart failure and changes in insulin and IGF1 levels. Whether human cardiac microvascular endothelial cells (HMVEC-Cs) are sensitive to insulin and/or IGF1 is not known. We studied the role of insulin receptors (IRs) and IGF1 receptors (IGF1Rs) in metabolic, mitogenic and anti-inflammatory responses to insulin and IGF1 in HMVEC-Cs and human umbilical vein endothelial cells (HUVECs). IR and IGF1R gene expression was studied using real-time RT-PCR. Receptor protein expression and phosphorylation were determined by western blot and ELISA. Metabolic and mitogenic effects were measured as glucose accumulation and thymidine incorporation. An E-selectin ELISA was used to investigate inflammatory responses. According to gene expression and protein in HMVEC-Cs and HUVECs, IGF1R is more abundant than IR. Immunoprecipitation with anti-IGF1R antibody and immunoblotting with anti-IR antibody and vice versa, showed insulin/IGF1 hybrid receptors in HMVEC-Cs. IGF1 at a concentration of 10−8 mol/l significantly stimulated phosphorylation of both IGF1R and IR in HMVEC-Cs. In HUVECs IGF1 10−8 mol/l phosphorylated IGF1R. IGF1 stimulated DNA synthesis at 10−8 mol/l and glucose accumulation at 10−7 mol/l in HMVEC-Cs. TNF-α dramatically increased E-selectin expression, but no inflammatory or anti-inflammatory effects of insulin, IGF1 or high glucose were seen. We conclude that HMVEC-Cs express more IGF1Rs than IRs, and mainly react to IGF1 due to the predominance of IGF1Rs and insulin/IGF1 hybrid receptors. TNF-α has a pronounced pro-inflammatory effect in HMVEC-Cs, which is not counteracted by insulin or IGF1.

Abstract 40: SARS-CoV-2/ACE2 Induces Vascular Inflammatory Responses In Human Microvascular Endothelial Cells Independently Of Viral Replication

Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
Augusto C Montezano ◽  
Livia Camargo ◽  
Sheon Mary ◽  
Karla B Neves ◽  
Francisco J Rios ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Viral Replication ◽  
Viral Infection ◽  
Protein Identification ◽  
Inflammatory Responses ◽  
Vascular Inflammation ◽  
Plaque Assay ◽  
Microvascular Endothelial Cells ◽  
Label Free ◽  
Microvascular Endothelial

SARS-CoV-2, the virus responsible for COVID19, binds to ACE2, via its spike protein S1 subunit, leading to viral infection and respiratory disease. COVID-19 is associated with cardiovascular disease and systemic inflammation. Since ACE2 is expressed in vascular cells we questioned whether SARS-CoV-2 induces vascular inflammation and whether this is related to viral infection. Human microvascular endothelial cells (EC) were exposed to recombinant S1p (rS1p) 0.66 μg/mL for 10 min, 5h and 24h. Gene expression was assessed by RT-PCR and levels of IL6 and MCP1, as well as ACE2 activity, were assessed by ELISA. Expression of ICAM1 and PAI1 was assessed by immunoblotting. ACE2 activity was blocked by MLN4760 (ACE2 inhibitor) and siRNA. Viral infection was assessed by exposing Vero E6 (kidney epithelial cells; pos ctl) and EC to 10 5 pfu of SARS-CoV-2 where virus titre was measured by plaque assay. Co-IP coupled mass spectrometry protein identification and label free proteomics were used to investigate ACE2-mediated signalling. rS1p increased IL6 mRNA (14.2±2.1 vs. C:0.61±0.03 2^-ddCT) and levels (1221.2±18.3 vs. C:22.77±3.2 pg/mL); MCP1 mRNA (5.55±0.62 vs. C:0.65±0.04 2^-ddCT) and levels (1110±13.33 vs. C:876.9±33.4 pg/mL); ICAM1 (17.7±3.1 vs. C:3.9±0.4 AU) and PAI1 (5.6±0.7 vs. C: 2.9±0.2), p<0.05. MLN4760, but not rS1p, decreased ACE2 activity (367.4±18 vs. C: 1011±268 RFU, p<0.05) and blocked rS1p effects on ICAM1 and PAI1. ACE2 siRNA blocked rS1p-induced IL6 release, ICAM1, and PAI1 responses as well as rS1p-induced NFκB activation. Proteomics analysis of the global effect of rS1, identified biological process enrichment of proteins from virus transcription and NFκB signalling. ACE2 Co-IP identified 216 interacting proteins (filtered with ≥1 unique peptide, 1% FDR), linked to cytokine production and inflammation. EC were not susceptible to SARS-CoV-2 infection, while the virus replicated well in Vero E6. In conclusion, we demonstrate that rS1p induces an inflammatory response through ACE2 in endothelial cells. These effects seem to be independent of viral infection. Our findings suggest that vascular inflammation in COVID-19 involves activation of ACE2-mediated pro-inflammatory signalling that may be unrelated to viral replication.

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